Preparation method and application of oral microspheres containing chlorella and ginseng polysaccharide

Abstract

The invention discloses a preparation method and application of oral microspheres containing chlorella and ginseng polysaccharide, and belongs to the technical field of biological medicine. The oral microspheres are prepared in a water-in-oil manner by utilizing a microfluidic technology, the microspheres are loaded with chlorella and ginseng polysaccharide, and a thermal response gel network is formed by taking methylacryloylated gelatin as a carrier. The prepared microspheres are stable in the stomach environment, can be gelated and gradually degraded under the induction of temperature in the intestinal tract after being orally taken, and slowly release active ingredients, so that the intestinal targeted delivery efficiency of the microspheres is effectively improved. The oral microspheres have multiple functions of regulating intestinal flora, repairing intestinal barriers, reducing intestinal permeability and regulating system immunity, and can remarkably improve intestinal and joint pathological manifestations related to rheumatoid arthritis. Furthermore, the joint cavity injection glucocorticoid is combined, so that the synergistic treatment of intestinal tracts and joints can be realized, and the anti-inflammatory and tissue protection effects are enhanced. The system is good in safety and stable in preparation process, and has wide application potential in rheumatoid arthritis and other chronic inflammatory diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing oral microspheres containing Chlorella and ginseng polysaccharides and their application. Background Technology

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease whose pathogenesis is closely related to gut microbiota dysbiosis and barrier dysfunction, forming a pathological link in the "gut-joint axis." Increased intestinal permeability leads to the entry of microbial metabolites into the circulation, triggering systemic immune inflammation and exacerbating joint damage.

[0003] Current mainstream treatments for rheumatoid arthritis (RA) rely on drugs such as glucocorticoids. While these can control joint symptoms, long-term use leads to significant adverse reactions and fails to effectively correct the key pathological link of gut microbiota imbalance. Chlorella vulgaris and ginseng polysaccharides, as natural active substances, have the potential to regulate gut microbiota, enhance barrier function, and exert systemic anti-inflammatory effects, offering new insights for RA management. However, oral administration of these two active ingredients faces significant challenges: they are easily degraded and inactivated in the acidic environment of the stomach; they are difficult to target and effectively accumulate in the colon; and their lack of intestinal retention capacity results in a short duration of action. Existing oral formulation technologies cannot provide effective gastrointestinal protection and colon-targeted delivery, leading to low bioavailability and severely limiting their clinical application.

[0004] Therefore, developing a novel oral delivery system that can protect the release of active ingredients through the stomach, target the colon, and prolong the intestinal action time is of great clinical significance and application value. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a method for preparing oral microspheres containing Chlorella and ginseng polysaccharides, and their applications. This microsphere system utilizes a thermosensitive gelatin methacryloyl (GelMA) hydrogel as a carrier, prepared via microfluidic technology. The synergistic loading of Chlorella and ginseng polysaccharides effectively protects them from degradation in the gastric environment and enables responsive release in the intestine, thereby improving the oral bioavailability of the drug. The prepared oral microspheres enhance the retention time and bioavailability of Chlorella and ginseng polysaccharides in the intestine, effectively repairing intestinal barrier function, regulating gut microbiota composition, reducing inflammation levels, and can be used in combination with locally injected glucocorticoids to alleviate RA joint symptoms while achieving systemic immune regulation and maintaining intestinal homeostasis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing oral microspheres containing Chlorella and ginseng polysaccharides, the specific steps of which are as follows: A mixture of gelatin methacryloyl (GelMA), the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), Chlorella vulgaris, and ginseng polysaccharides was used as the aqueous phase, and sorbitan oleate (Span80) and paraffin oil were used as the oil phase. The aqueous and oil phases were simultaneously injected into the microfluidic chip at a flow rate ratio of 1:8, forming stable monodisperse droplets. These droplets were then subjected to ultraviolet light (365 nm, 80 mW / cm²). 2 After being irradiated for several seconds, the solidification process is completed, resulting in stable oral microspheres with thermosensitive properties.

[0007] Further, the aqueous phase was prepared as follows: gelatin methacrylamide (GelMA) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) were dissolved in 1x phosphate-buffered saline (PBS) to prepare a homogeneous solution, wherein the mass concentration of GelMA was 0.25% (w / v) and the mass concentration of LAP was 0.25% (w / v). Subsequently, Chlorella cells and ginseng polysaccharide (GPS) were added to the solution to achieve a final Chlorella cell density of approximately 1.4 × 10⁻⁶. 7 The final concentration of GPS was 2 mg / mL. The oil phase consisted of Span 80 and paraffin oil in a mass ratio of 1:9 and was preheated at 40°C. The final microspheres had an average particle size of 60–64 μm.

[0008] This invention also provides an application of oral microspheres containing Chlorella and ginseng polysaccharides obtained by the above preparation method, wherein the microspheres have one or more of the following uses: 1) Preparation of an oral drug delivery system for adjuvant treatment of rheumatoid arthritis; 2) To prepare oral medications for repairing the intestinal epithelial barrier and / or reducing intestinal permeability; 3) Prepare microecological regulators for regulating the structure of intestinal flora and improving the intestinal microenvironment; 4) To prepare an adjuvant formulation for use in combination with the glucocorticoid trifluorochlorothiazide in the treatment of rheumatoid arthritis; 5) As an adjunct therapy for systemic inflammatory diseases caused by intestinal flora imbalance.

[0009] The CG@GelMA thermosensitive oral microspheres constructed in this invention are based on natural active ingredients, have good ingredient safety, raw material availability and process scalability, are suitable for large-scale production and have good clinical translation potential.

[0010] The present invention has the following beneficial effects: The resulting microspheres exhibit a dense structure and excellent thermal responsiveness, remaining stable in the stomach. After passing through the stomach intact, they undergo slow hydrolysis in the small intestine, releasing the active ingredients and significantly improving the oral bioavailability of Chlorella and ginseng polysaccharides. Gelatin methacrylamide (GelMA) serves as the carrier matrix, endowing the microspheres with excellent controlled-release properties and biodegradability. Functionally, Chlorella (CV) and ginseng polysaccharides (GPS) work synergistically within the microspheres, enhancing intestinal epithelial barrier function, promoting the expression of tight junction proteins (Claudin-1, Occludin, ZO-1), regulating intestinal flora composition, and promoting the production of short-chain fatty acids, thereby reducing intestinal inflammation and oxidative stress. This reduces inflammatory leakage and corrects the immune imbalance associated with rheumatoid arthritis. Furthermore, the chlorophyll contained in Chlorella possesses natural red fluorescence, enabling non-invasive imaging and dynamic tracking of the gastrointestinal tract via oral administration without the need for exogenous dye labeling. This oral system can independently improve RA symptoms and exhibits a synergistic effect when combined with local injection of glucocorticoids (trifluorochlorothiazol), significantly relieving joint inflammation, improving bone and joint structure, and enhancing athletic performance. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the microfluidic preparation process of the thermosensitive oral microspheres containing Chlorella and ginseng polysaccharides based on thermosensitive gelatin methacrylamide as described in this invention; Figure 2 The images show the structural characterization of CG@GelMA microspheres, where image a is a scanning electron microscope image, image b is a bright-field optical microscope image, and image c is a fluorescence microscope image. Figure 3 The figures show the release curves of ginseng polysaccharides from CG@GelMA microspheres in artificial gastric fluid, artificial small intestinal fluid, and artificial colonic fluid. Figure a shows the release curves of ginseng polysaccharides in artificial gastric fluid and artificial small intestinal fluid, and Figure b shows the release curve of ginseng polysaccharides in artificial colonic fluid. Figure 4 The fluorescence distribution of different organs in vitro after mice were orally administered CG@GelMA, under the FITC channel and the autofluorescence channel of Chlorella. Figure 5 Scanning electron micrographs showing the morphological changes of CG@GelMA microspheres in the stomach, ileum, cecum, and colon after oral administration; Figure 6 The images show the efficacy evaluation of collagen-induced arthritis mouse models after treatment with CG@GelMA, TAA, or a combination of both. Image a is a Micro-CT image, and image b is an image of joint HE, TRAP, and Safranin O / Fast Green staining. Figure 7Immunofluorescence images of tight junction protein expression in the ileum and colon of CIA mice in different treatment groups; where figure a is the immunofluorescence image of tight junction protein expression in the ileum of mice, and figure b is the immunofluorescence image of tight junction protein expression in the colon of mice. Figure 8 This is a biocomparison chart of the long-term oral administration of CG@GelMA, including HE sections of major organs and comparisons of blood routine, blood biochemical indicators (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; BUN, blood urea nitrogen; CREA, serum creatinine). Figure a shows HE sections of major organs: heart, liver, spleen, lung, kidney, stomach, and intestine. Figure b shows the comparison of blood routine, blood biochemical indicator, and blood biochemical indicator results. Detailed Implementation

[0012] 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.

[0013] Example 1 Preparation method of CG@GelMA microspheres.

[0014] Gelatin methacrylamide (GelMA) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) were co-dissolved in phosphate-buffered saline (PBS) to prepare a homogeneous solution with a GelMA concentration of 0.25% (w / v) and a LAP concentration of 0.25% (w / v). Subsequently, approximately 7 × 10⁻⁶ g of [amount missing] ... 7 One Chlorella vulgaris cell was thoroughly mixed with 10 mg of ginseng polysaccharide (GPS) to achieve a concentration of approximately 1.4 × 10⁻⁶ in the final aqueous solution (total volume approximately 5 mL). 7 The Chlorella vulgaris cells were purchased from Shanghai Guangyu Biotechnology Co., Ltd., and the ginseng polysaccharide (GPS) was purchased from Shanghai Maclean Biotechnology Co., Ltd. This mixed solution served as the aqueous phase in the microfluidic preparation process. The oil phase consisted of Span 80 and paraffin oil in a mass ratio of 1:9, and was preheated to 40°C.

[0015] In the microfluidic fabrication process, an aqueous phase and an oil phase were simultaneously injected into a microfluidic chip at a flow rate ratio of 1:8 using a precision pump, generating monodisperse droplets through shear force. The microfluidic chip used was a PDMS chip, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. Throughout the process, the system was maintained at 37°C using a heating lamp to preserve the low viscosity of the GelMA. After collecting the generated droplets, they were immediately subjected to microfluidic analysis at 365 nm and 80 mW / cm². 2 Crosslinking and curing under ultraviolet light resulted in structurally stable microspheres. The final product was thoroughly washed with 1×PBS to remove residual oil phase. The obtained CG@GelMA microspheres were spherical, and size distribution analysis showed that the microsphere diameter ranged from 56 to 76 μm, with the majority (38.5%) falling within the 60-64 μm range. A detailed preparation diagram is shown below. Figure 1 The structural characterization results are shown in Figure 2 .

[0016] Example 2 In vitro drug release performance of CG@GelMA.

[0017] The in vitro release behavior of ginseng polysaccharide (GPS) in CG@GelMA was determined using the dialysis bag method at 37℃. Five ml of CG@GelMA sample containing GPS was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa. The sample was then sequentially immersed in 15 mL of simulated gastric fluid (SGF, Yuanye Biotechnology, Shanghai, China) for 2 hours, followed by 15 mL of simulated intestinal fluid (SIF, Yuanye Biotechnology) for 6 hours, and finally 15 mL of simulated colonic fluid (SCF, Yuanye Biotechnology) for 72 hours. At different time points (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72 h), 1 mL of the release medium was collected and immediately replenished with an equal volume of the corresponding simulated liquid of the same type. The absorbance of the release medium was measured at 282 nm using a UV-2600 UV-Vis spectrophotometer, and the cumulative release rate was calculated based on standard curves generated by GPS in SGF, SIF, and SCF, respectively. The results showed that the microspheres released the drug slowly in gastric fluid (<25% release in 2 h), gradually increased in intestinal fluid, and significantly increased in colonic fluid, reaching a cumulative release rate of 71.8% in 72 h. The release curves are shown below. Figure 3 As shown in a and 3b.

[0018] Example 3 CG@GelMA's ability to detect organ distribution in vitro and in vivo.

[0019] To investigate the gastrointestinal transport and distribution of CG@GelMA microspheres in vivo, female Balb / c nude mice (6 weeks old) were used. After fasting overnight, the mice were administered 300 μL of CG@GelMA microsphere suspension (with a CV concentration of 1.4 × 10⁻⁶) orally by gavage. 7 (cells / mL, GPS at 2 mg / mL). For ease of fluorescence tracking, GPS was labeled with FITC. Whole-body fluorescence imaging of mice under isoflurane anesthesia was performed at predetermined time points after drug administration (0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48 h) using an IVIS Lumina LT Series III in vivo imaging system (PerkinElmer, USA). Mice were sacrificed at specific time points (n=3 at each time point), and ex vivo fluorescence imaging of major organs (heart, liver, spleen, lung, kidney) and gastrointestinal segments (stomach, small intestine, cecum, colon) was collected. Both in vivo and ex vivo fluorescence images were quantitatively analyzed using Living Image 4.5 software.

[0020] Figure 4 The red (CV autofluorescence) / green (FITC-GPS) fluorescence distribution in major digestive organs of mice after oral administration of CG@GelMA was demonstrated. From 0.5 to 2 hours post-oral gavage, the fluorescence signal was mainly enriched in the stomach and upper small intestine, indicating that the microspheres were undergoing gastric emptying and small intestinal transit during this stage. Subsequently, from 3 to 8 hours, the signal center shifted to the cecal region, and significant fluorescence was detected in the colon, confirming that most microspheres had been successfully delivered to the colon. From 10 hours onwards, the overall fluorescence signal in the gastrointestinal region began to decrease significantly, becoming virtually undetectable by 48 hours. These results indicate that the microspheres can remain in the intestine for a long period without significant distribution throughout the body.

[0021] Example 4 Degradation status in vivo.

[0022] Mice were fasted overnight and then administered 300 μL of CG@GelMA microsphere suspension (with a CV concentration of 1.4 × 10⁻⁶) orally by gavage. 7 Cells / mL (GPS: 2 mg / mL), after 2 hours, the cells were sacrificed and the contents of the stomach, ileum, cecum, and colon were collected. The contents were appropriately diluted with 1×PBS. One portion was observed for microsphere morphology using an optical microscope; the other portion was fixed, dehydrated, critically dried, and sputter-coated with gold, and then the surface morphology and degradation of the microspheres were observed using a scanning electron microscope (SEM). Figure 5 As shown, the microspheres maintain a relatively intact spherical structure in the stomach, begin to swell and erode on the surface after entering the small intestine, and show significant structural disintegration and almost complete degradation in the colon, confirming their colon-specific release characteristics.

[0023] Example 5 Protective effect against CTLA-4 and PD-1 monoclonal antibody-induced colitis.

[0024] The efficacy of treatment was evaluated using a collagen-induced arthritis (CIA) model. Male DBA / 1 mice (6–8 weeks old) were intradermally immunized with 100 μg bovine type II collagen (emulsified in complete Freund's adjuvant) at the base of the tail on day 0, and boosted with collagen emulsified incomplete Freund's adjuvant on day 21. On day 25, mice (n=20) that developed arthritis were randomly divided into four groups: (i) control group (oral administration of ddH2O + intra-articular injection of PBS); (ii) CG@GelMA group (oral administration of CG@GelMA daily, 300 μL / mouse, containing CV 1.4 × 10⁻⁶). 7 (iii) TAA group (oral ddH2O + intra-articular injection of triamcinolone acetonide on days 30, 36, 42 and 48, 5 μL / time, concentration 2 mg / mL); (iv) CG@GelMA + TAA combined treatment group (receiving both of the above treatment regimens simultaneously). Figure 6 Figure a shows that the combined treatment group exhibited good preservation of bone and joint structures in Micro-CT imaging. Figure 6 b HE, TRAP, and Safranin O / Fast Green staining results also showed that it significantly reduced inflammation, inhibited osteoclasts, and repaired cartilage, with the combination group being superior to single therapy.

[0025] Example 6 It improves intestinal tight junctions and barrier function.

[0026] Ileal and colonic tissues were collected from mice in each group of Experiment 5. A portion of the tissue was fixed, embedded in paraffin, sectioned, and subjected to immunofluorescence staining. Primary antibodies against Claudin-1, Occludin, and ZO-1 (Servicebio, 1:100 dilution) and corresponding secondary fluorescent antibodies were used. After nuclear counterstaining with DAPI, images were acquired using a Zeiss LSM 880 confocal microscope, and fluorescence intensity was quantitatively analyzed using ImageJ software. Results are as follows: Figure 7 As shown in a and 7b, the combined expression of CG@GelMA + TAA histones was the strongest, indicating that it significantly improved intestinal barrier function.

[0027] Example 7 Biosafety assessment of long-term oral administration of CG@GelMA.

[0028] To evaluate the safety of long-term oral administration of CG@GelMA, the following groups were established: CG@GelMA group, CV alone group, GPS alone group, blank GelMA microsphere group, and blank control group (PBS). Mice in each group (n=5) were administered the corresponding formulation (300 μL) by gavage daily for 30 consecutive days. After the administration, blood and major organs were collected from the mice, and hematoxylin and eosin (HE) sections were prepared for complete blood count and blood biochemical analysis. The assays included white blood cells, red blood cells, hemoglobin, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), platelets, hematocrit, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine. Figure 8 No abnormal pathological findings were observed in the HE sections of the major organs. Figure 8 b shows that there are no significant differences in blood routine and blood biochemical indicators among the groups of mice, indicating that the present invention has good oral safety.

Claims

1. A method for preparing oral microspheres containing Chlorella and ginseng polysaccharides, characterized in that, The specific steps are as follows: A mixture of gelatin methacrylamide, photoinitiator, Chlorella vulgaris and ginseng polysaccharide was used as the aqueous phase. The aqueous phase was heated to 37°C and kept at that temperature. It was then simultaneously injected into a microfluidic chip with an oil phase preheated to 40°C at a flow rate ratio of 1:

8. The mixture was emulsified to form stable monodisperse spherical droplets. The droplets were collected and cross-linked and cured by ultraviolet light to obtain stable oral microspheres with thermosensitive properties. The aqueous phase preparation process involved dissolving gelatin methacrylamide and a photoinitiator in 1xPBS to prepare a 1xPBS solution containing 0.25% w / v gelatin methacrylamide and 0.25% w / v photoinitiator. Chlorella cells and ginseng polysaccharides were then added to the solution, bringing the final concentration of Chlorella cells to 1.4 × 10⁻⁶. 7 The final concentration of ginseng polysaccharide was 2 mg / mL.

2. The preparation method according to claim 1, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

3. The preparation method according to claim 1, characterized in that, The oil phase consists of Span 80 and paraffin oil in a mass ratio of 1:

9.

4. The preparation method according to claim 1, characterized in that, The curing step is performed at a wavelength of 365 nm and a temperature of 80 mW / cm². 2 Irradiate under ultraviolet light for 5 minutes.

5. An oral microsphere containing Chlorella and ginseng polysaccharide, obtained by the preparation method of claim 1.

6. The oral microspheres according to claim 5, characterized in that, The average particle size of the oral microspheres is 60–64 μm.

7. An application of the oral microspheres according to claim 5, characterized in that, The applications include one or more of the following: 1) Preparation of an oral drug delivery system for adjuvant treatment of rheumatoid arthritis; 2) To prepare oral medications for repairing the intestinal epithelial barrier and / or reducing intestinal permeability; 3) Prepare microecological regulators for regulating the structure of intestinal flora and improving the intestinal microenvironment; 4) To prepare an adjuvant formulation for use in combination with the glucocorticoid trifluorochlorothiazide in the treatment of rheumatoid arthritis; 5) As an adjunct therapy for systemic inflammatory diseases caused by intestinal flora imbalance.

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