An enteric-targeted controlled-release microalgae pharmaceutical composition, preparation method and application

Abstract

The application discloses an intestinal-targeted controlled-release microalgae pharmaceutical composition, which comprises a hydrophobic drug, a micellar material and microalgae, wherein the hydrophobic drug is first loaded on the micellar material to form a drug-loaded micelle, the solubility of the hydrophobic drug is improved, and the drug-loaded micelle solution penetrates into the interior of the microalgae cells through a passive water absorption process of dried microalgae instead of being attached to the surface of the microalgae. The natural cell wall of the microalgae itself is solid and acid-resistant, which is used as a first physical barrier to prevent the release of the hydrophobic drug in a gastric acid environment, and the effective concentration and residence time of the hydrophobic drug in the intestinal action site are significantly improved, so that the treatment effect is better, and the intestinal disease and kidney disease drug can be prepared.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to an intestinal-targeted controlled-release microalgae drug composition, its preparation method, and its application. Background Technology

[0002] Budesonide, chemically named 16α,17α-(22R,S)-propylmethylenedioxy-pregn-1,4-diene-11β,21-dihydroxy-3,20-dione, has the molecular formula C1 25 H 34 O6, with a molecular weight of 430.5, has the following structural formula.

[0003]

[0004] Budesonide is a glucocorticoid primarily used for anti-inflammatory and immunomodulatory purposes. Its indications cover multiple systemic diseases, and it is often administered orally for the treatment of inflammatory bowel disease and primary immunoglobulin A (IgA) nephropathy. Budesonide is a drug with low solubility and high permeability; its absorption is mainly limited by the drug's own solubility. The solubility of budesonide is affected by the pH of the dissolution medium, increasing as the pH decreases. Therefore, after oral administration, budesonide is absorbed more readily due to its high solubility in the stomach and less readily due to its low solubility in the intestines.

[0005] However, in treating inflammatory bowel disease and primary immunoglobulin A (IgA) nephropathy, neither drug acts in the stomach. The lesions in inflammatory bowel disease are distributed in the ileum, colon, and cecum, while in IgA nephropathy, the drug primarily acts on the Peyer's lymph nodes in the terminal ileum. Direct oral administration of budesonide results in the drug dissolving and being absorbed mainly in the stomach, potentially leading to systemic toxicity. In the target intestinal segment, drug dissolution and absorption are less efficient, hindering its therapeutic effect.

[0006] To address this issue, researchers have developed enteric-coating strategies for budesonide. For example, Chinese patent publication CN118591376A uses enteric-coating polymer materials to prevent drug release in gastric juice and release it in intestinal juice after 2 hours. Chinese patent publication CN115554246A uses an amorphous method to increase budesonide solubility and employs hydroxypropyl methylcellulose acetate succinate for enteric coating. Chinese patent publication CN117157079A uses hydroxypropyl methylcellulose as the microparticle material. While all the aforementioned patents achieve effective enteric coating, they all use chemically synthesized polymers as materials for intestinal release, and the processes for colonic release are relatively complex.

[0007] Microalgae, as natural biomaterials, have significant advantages in the development of novel drug delivery systems due to their excellent biocompatibility and biodegradability. However, published reports often utilize the electronegativity of the microalgal cell wall surface to allow both drugs and gel materials to adhere to its surface, while the various natural channels and internal structures within the cell wall are not fully utilized for drug delivery.

[0008] For example, patent (CN119925260A) discloses a method for preparing a colon-targeted common chlorella / paeoniflorin hydrogel. In this method, both the drug and the hydrogel are on the outside of the chlorella, and the common chlorella is only used as a nutrient containing polysaccharides and proteins to regulate gastrointestinal function, without fully utilizing its drug delivery advantages. Patent (CN119236091A) discloses a chlorella-based meibomian gland dysfunction treatment composition. Although dexamethasone is loaded onto chlorella as a carrier, the drug is adsorbed on the surface of the chlorella, and its drug loading efficiency is significantly limited by the surface charge characteristics of the algae and external environmental parameters (such as pH value and ionic strength), and the carrier advantages of chlorella are not fully reflected.

[0009] Therefore, there is an urgent need to find a microalgae drug composition that can fully utilize the natural structure and carrier advantages of microalgae to achieve targeted delivery and intestinal sustained release. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides an intestinal-targeted controlled-release microalgae drug composition comprising a hydrophobic drug, a micelle material, and microalgae, wherein the hydrophobic drug is loaded onto the micelle material to form drug-loaded micelles, and the drug-loaded micelles are located inside the microalgae.

[0011] The lipid-water partition coefficient (logP) of the hydrophobic drug is 2 to 4.

[0012] Existing microalgae drug compositions primarily rely on the adsorption of drugs onto the surface of microalgae by surface charges, allowing the drugs to be easily released in the acidic environment of the stomach. However, the intestinal-targeted controlled-release microalgae drug composition described in this application first loads a hydrophobic drug onto a micelle material to form drug-loaded micelles, improving the solubility of the hydrophobic drug. Then, through a passive water absorption process during drying of the microalgae, the drug-loaded micelle solution permeates into the microalgae cells, rather than adhering to the surface. This microalgae drug composition significantly increases the effective concentration and retention time of the hydrophobic drug at the site of action in the intestine.

[0013] The microalgae drug composition of the present invention utilizes the strong and acid-resistant natural cell wall of microalgae to slow down the release of hydrophobic drugs in the acidic environment of the stomach. When the microalgae carrier enters the intestine, its cell wall is gradually degraded and destroyed by digestive enzymes (such as cellulase and pectinase) or bile salts secreted by intestinal microorganisms, thereby achieving targeted intestinal release of hydrophobic drugs.

[0014] Preferably, in the intestinal-targeted controlled-release microalgae drug composition, the mass ratio of the hydrophobic drug to the micelle material is 1:10~100; the mass ratio of the hydrophobic drug to the microalgae is 1:50~1000.

[0015] More preferably, the mass ratio of the hydrophobic drug to the micelle material is 1:40~60; the mass ratio of the hydrophobic drug to the microalgae is 1:400~600.

[0016] Preferably, the hydrophobic drug is one or more of budesonide, Nile Red dye, resveratrol, curcumin, quercetin, and green tea polyphenols.

[0017] In this invention, budesonide (logP=3.14), Nile red dye (logP=3.65), resveratrol (logP=3.14), curcumin (logP=2.85), quercetin (logP=2.08), and green tea polyphenols (logP=2.08) all have lipid-water partition coefficients in the range of 2 to 4 and are all hydrophobic drugs. They can be loaded onto micelle materials to form drug-loaded micelles, thereby improving their solubility. At the same time, the drug-loaded micelle solution is allowed to penetrate into the microalgal cells through the passive water absorption process of dried microalgae, forming a microalgal drug composition with an outer layer of microalgae and an inner layer of drug-loaded micelles.

[0018] Preferably, the micelle material is one or more of nonionic surfactants, amphoteric surfactants, and anionic surfactants.

[0019] In this invention, the nonionic surfactant can be one or more of the following: caprylic / capric acid glyceride, sorbitan monolaurate, sorbitan monopalmitate, polysorbate 20, polysorbate 40, polysorbate 80, polyoxyethylene fatty alcohol ether, polyoxyethylene alkylphenol ether, polyoxyethylene fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene oleic acid glyceride, polyoxyethylene linoleic acid glyceride, vitamin E polyethylene glycol succinate, polyethylene glycol 15-hydroxystearate, polyoxypropylene ethylene oxide glycerol ether, gum arabic, gelatin, saponins, and cholesterol.

[0020] The amphoteric surfactant can be one or more of the following: cocamidopropyl betaine, lauramidopropyl betaine, oleamidopropyl betaine, dodecyl betaine, cocamidopropyl hydroxysulfonyl betaine, lauramidopropyl hydroxysulfonyl betaine, tetradecyl sulfonyl betaine, sodium lauroyl sarcosinate, disodium oleoamphodiacetate, disodium laurylamphodiacetate, lauryl dimethylamine oxide, cocamidopropyl dimethylamine oxide, and lecithin.

[0021] Anionic surfactants can be one or more of sodium stearate, sodium dioctyl succinate, sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium alginate, sodium docusate, cholate, and sodium deoxycholate.

[0022] More preferably, the hydrophobic drug is budesonide or Nile Red dye, and the micelle material is a nonionic surfactant.

[0023] Nonionic surfactants do not have a net charge, and their interaction with the surface of microalgae is not affected by electrostatic forces. They mainly rely on hydrophobic interactions and steric effects, which can more effectively penetrate the cell barrier and promote the entry of micelles loaded with budesonide or Nile Red dye into the interior of microalgae, thereby improving drug loading efficiency and avoiding aggregation or surface adsorption problems caused by charge.

[0024] Preferably, the particle size of the drug-loaded micelles is 10~1200 nm.

[0025] Preferably, the microalgae is one or more of Spirulina, Chlorella, Chlamydomonas reinhardtii, and Pseudomicroalgae.

[0026] More preferably, the hydrophobic drug is budesonide or Nile Red dye, the micelle material is a nonionic surfactant, and the microalgae is Chlorella vulgaris.

[0027] More preferably, the hydrophobic drug is budesonide or Nile Red dye, the micelle material is octanoic acid-capric acid-decanoic acid polyethylene glycol glycerol ester, and the microalgae is Chlorella proteoglycans.

[0028] Preferably, the intestinal-targeted controlled-release microalgae drug composition further includes a gelling material, which is one or more of the following: polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus®), gelatin, collagen, hyaluronic acid, alginate, chitosan, agarose, polyacrylamide, poloxamer, polyvinyl alcohol, polyethylene glycol, and polymethyl methacrylate.

[0029] More preferably, the mass ratio of the hydrophobic drug to the gel material is 1:20~400.

[0030] More preferably, the intestinal-targeted controlled-release microalgae drug composition also contains a metal inorganic salt, wherein the metal inorganic salt is one or more of sodium chloride, potassium chloride, calcium chloride, sodium bromide, potassium bromide, and calcium bromide.

[0031] In this invention, by adding a temperature-responsive gel material and a metallic inorganic salt, a micelle liquid containing a temperature-sensitive gel is formed under low-temperature conditions. After being absorbed into the microalgae, it forms a gel at body temperature. This improvement prolongs the release time of hydrophobic drugs, allowing them to reach the ileum, cecum, and colon, achieving sustained intestinal release.

[0032] More preferably, the mass ratio of the hydrophobic drug to the inorganic metal salt is 1:0.1~100.

[0033] More preferably, the gel material is a polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and the metal inorganic salt is sodium chloride.

[0034] The microalgae drug composition of this invention, upon the addition of a temperature-responsive gelling material, allows the gelling material, micelle material, and hydrophobic drug to enter the microalgae in a liquid state at low temperatures, forming a gel at near body temperature. The addition of the temperature-responsive gelling material further prolongs the drug release time and increases the effective concentration and retention time of the hydrophobic drug at the intestinal site of action. The addition of a metal inorganic salt allows for the regulation of the gel-forming state and temperature of the temperature-sensitive gel.

[0035] The present invention also provides a method for preparing the above-mentioned intestinal-targeted controlled-release microalgae drug composition, comprising the following steps:

[0036] Hydrophobic drugs, micelle materials, gel materials, and metal inorganic salts are dissolved in water to obtain a homogeneous solution. Dry microalgae powder is added to the homogeneous solution, and the mixture is stirred overnight. The surface-adsorbed hydrophobic drugs are washed away, and the mixture is centrifuged to obtain an intestinal-targeted controlled-release microalgae drug composition.

[0037] Preferably, the dried microalgae powder is obtained by freeze-drying, spray drying or hot air drying of microalgae or algal mud.

[0038] The present invention also provides the application of the above-mentioned intestinal-targeted controlled-release microalgae drug composition in the preparation of drugs for treating intestinal diseases and kidney diseases.

[0039] Preferably, the intestinal disease is functional dyspepsia, chronic enteritis, inflammatory bowel disease, intestinal cancer, or irritable bowel syndrome, and the kidney disease is primary immunoglobulin A (IgA) nephropathy.

[0040] The intestinal-targeted controlled-release microalgal drug composition prepared in this invention utilizes the protective effect of the outer layer of microalgal cells to reduce drug dissolution in gastric juice and achieve long-term release in the targeted intestine, making it particularly suitable for diseases that require drugs to exert their effects in the intestine.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] (1) Compared with traditional oral administration, the microalgae drug composition of the present invention can significantly improve the effective concentration and retention time of the drug at the intestinal site of action, thereby better exerting the therapeutic effect. It is also green and edible, easy to swallow orally, and low in cost.

[0043] (2) Hydrophobic drugs are formed into drug-loaded micelles with nanoparticle size. These micelles are passively absorbed by dried algal powder, allowing them to enter the microalgae through the pores on the microalgae surface. This improvement significantly increases the solubility of hydrophobic drugs and enables them to penetrate the microalgae rather than adhering to the surface. Furthermore, once inside the microalgae, the hydrophobic drugs are not released under gastric acid conditions, but only in the intestinal environment. This improvement reduces the degradation and absorption of hydrophobic drugs in the stomach, achieving targeted release into the intestinal tract.

[0044] (3) This invention utilizes a temperature-responsive gel material to form a micelle liquid containing a temperature-sensitive gel under low-temperature conditions. After being absorbed into the microalgae, it forms a gel under body temperature conditions. This improvement prolongs the release time of hydrophobic drugs, allowing them to reach the ileum, cecum, and colon, increasing the drug concentration in these intestinal segments and achieving sustained intestinal release.

[0045] (4) The budesonide and microalgae composition provided by the present invention has significant therapeutic advantages in treating a mouse colitis model induced by sodium dextran sulfate. Attached Figure Description

[0046] Figure 1 This is a flowchart illustrating the preparation process of the microalgae drug composition containing budesonide gel obtained in Example 23.

[0047] Figure 2 The diagrams show the state and water content of Chlorella proteoglycans. In the diagrams, A shows the morphology of Chlorella proteoglycans algal mud and algal powder, B shows the weight change of Chlorella proteoglycans before and after freeze-drying, and C shows the water content of Chlorella proteoglycans after reconstitution of the freeze-dried powder in the algal mud state.

[0048] Figure 3 The image shows the pore size distribution of Chlorella pyrenoidosa powder. In the image, A is the logarithmic plot of nitrogen adsorption isotherms, and B is the graph showing the relationship between pore volume and pore size.

[0049] Figure 4 These are fluorescence confocal images of the microalgae drug compositions containing Nile Red dye prepared in Examples 19-22.

[0050] Figure 5 The image shows fluorescence imaging of the microalgae drug composition containing Nile Red dye and Nile Red micelle solution prepared in Example 19 in the stomach and intestine of mice.

[0051] Figure 6The graph shows the Nile Red dye content in the stomach and intestines of mice in the microalgae drug composition and Nile Red micelle solution prepared in Example 19.

[0052] Figure 7 The in vitro release curves of budesonide suspension, the budesonide-containing microalgae drug composition prepared in Example 4, and the budesonide-containing microalgae drug composition prepared in Example 23 are shown.

[0053] Figure 8 Concentration statistics of budesonide suspension, budesonide-containing microalgae drug composition prepared in Example 4, and budesonide-containing microalgae drug composition prepared in Example 23 in plasma, stomach, ileum, cecum, and colon tissues after administration for 1, 2, 4, and 8 hours.

[0054] Figure 9 The following are evaluation charts of the therapeutic effects of the microalgae drug composition containing budesonide prepared in Example 4 and the microalgae drug composition containing budesonide gel prepared in Example 23 on mice with dextran sulfate-induced colitis. In the charts, A is a mouse colon, B is a statistical chart of mouse DAI score, C is a statistical chart of mouse colon length, D is a statistical chart of mouse colon mass-to-length ratio, E is the statistical result of spleen index; F is the statistical result of colon histopathological score; G is a typical HE staining image of colon tissue (black arrow: local ulcer; red arrow: inflammatory cell infiltration; blue arrow: extensive fibrous connective tissue hyperplasia and replacement; green arrow: intestinal gland dilatation; *, p value < 0.05; **, p value < 0.01). Detailed Implementation

[0055] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited to the following embodiments.

[0056] All raw materials used in this invention are commercially available.

[0057] Example 1

[0058] Take 20 mg of budesonide and 200 mg of caprylic / capric acid polyethylene glycol glycerol ester, add 5 mL of ethanol to dissolve them completely, then remove the ethanol using a rotary evaporator. Add the mixture to 10 mL of water and stir to obtain a clear and transparent budesonide micelle solution. Add 5.5 g of lyophilized Chlorella pulveratum powder to the budesonide micelle solution and stir overnight to allow the budesonide micelle solution to be fully absorbed into the algal powder. Add 6 mL of ethanol and vortex for 1 min to wash away the budesonide micelle solution adsorbed on the surface of Chlorella pulveratum. Centrifuge at 5000 rpm for 10 min, discard the supernatant, and take out the lower solid material. Repeat the washing step twice. Finally, dry the lower solid material under reduced pressure at room temperature for 12 h to obtain a microalgal drug composition containing budesonide.

[0059] Examples 2-15

[0060] The preparation method is the same as in Example 1, except that the type and amount of micelle material are different, as shown in the table below.

[0061] Table 1: Differences between the budesonide-containing microalgal drug compositions in Examples 1-15

[0062]

[0063] Examples 16-18

[0064] The preparation method is the same as in Example 1, except that the species of microalgae are different, as shown in the table below.

[0065] Table 2: Differences between the budesonide-containing microalgal drug compositions prepared in Examples 1, 16-18

[0066]

[0067] Example 19

[0068] Weigh 20 mg of Nile Red dye and 1 g of octanoic acid-capric acid-polyethylene glycol glycerol ester. Stir them thoroughly to obtain a clear solution. Add the above solution to 10 mL of water and stir to obtain a clear and transparent Nile Red micelle solution. Add 5.5 g of dried Chlorella pulveratum powder to the Nile Red micelle solution and stir overnight to allow the Nile Red micelle solution to be fully absorbed into the algal powder. Add 6 mL of ethanol and vortex for 1 min to wash away the Nile Red dye adsorbed on the surface of Chlorella pulveratum. Centrifuge at 5000 rpm for 10 min, discard the supernatant, and take out the lower solid material. Repeat the washing step twice. The final lower solid material is dried under reduced pressure at room temperature for 12 h to obtain a microalgae drug composition containing Nile Red dye.

[0069] Examples 20-22

[0070] The preparation method is the same as in Example 19, except that the type and amount of micelle material are different, as shown in the table below.

[0071] Table 3: Differences between the microalgae drug compositions containing Nile Red dye prepared in Examples 20-22

[0072]

[0073] Example 23

[0074] Preparation process as follows Figure 1 As shown.

[0075] Weigh 20 mg of budesonide and 1 g of PEG-1,4-diethyl-caprylic acid and capric acid, and stir them thoroughly to obtain a clear solution. Then weigh 4.75 g of Soluplus® and 171 mg of sodium chloride, add them to 14.25 mL of water, stir at 4 °C for 4 h, and then let it stand at 4 °C for 12 hours to swell, thus preparing a gel solution.

[0076] The above solutions were mixed and stirred vigorously at 4 °C and 1500 rpm for 2 hours to obtain a homogeneous budesonide-gel-micelle solution. 10 g of dried Chlorella pulveratum powder was added to the above solution and stirred overnight to allow the budesonide-gel-micelle solution to be fully absorbed into the algal powder. 20 mL of ethanol was added, and the mixture was vortexed for 1 min to wash away the budesonide adsorbed on the surface of Chlorella pulveratum. The mixture was then centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the lower solid material was taken out and the washing step was repeated twice. The final lower solid material was dried under reduced pressure at room temperature for 12 h to obtain a microalgal drug composition containing budesonide gel.

[0077] Examples 24-38

[0078] The preparation method is the same as in Example 23, except that the mass ratio of budesonide, caprylic / capric acid glycerol, Soluplus®, sodium chloride, and Chlorella pulveratum powder is different, as shown in the table below.

[0079] Table 4: Differences between the microalgae drug compositions containing budesonide gel in Examples 23-38

[0080]

[0081] Examples 39-50

[0082] The preparation method is the same as in Example 23, except that the types of micelle materials, gel materials, metal inorganic salts or microalgae are different, as shown in the table below.

[0083] Table 5: Microalgae drug compositions containing budesonide gel in Examples 23, 39-50

[0084]

[0085] Experimental Analysis

[0086] Experimental Example 1: Determination of water content in Chlurella pyrenoidosa (CP)

[0087] Commercially available Chlorella pyrenoidosa algal sludge (source: Kangyuan Weike Direct Sales Store on Taobao) was used. The sludge was dispensed into 30-40g portions and placed in 50mL centrifuge tubes. The tubes were frozen at -80℃ for 8-12 hours. The frozen sludge was then transferred to a freeze dryer and freeze-dried at -60℃ for 48-72 hours. After drying, Chlorella pyrenoidosa algal powder (CP powder) was obtained. Figure 2 (A) The weight after freeze-drying is approximately 1 / 4 of the weight before freeze-drying. Figure 2 (B) In this case, the freeze-dried microalgae powder was reconstituted, and the Chlorella sample was dried in an oven at 105 ℃ to constant weight using the drying method. The moisture content was calculated by weighing. The moisture content of Chlorella sludge before freeze-drying (original state) and after reconstitution of freeze-dried algae powder were measured separately. The results are as follows: Figure 2 As shown in C, the water content of both groups remained at approximately 66%. After the freeze-dried algal powder was rehydrated and absorbed water, its water content could be restored to a level similar to that of the original algal mud, indicating that the freeze-drying process did not significantly damage the water absorption and retention capacity of Chlorella cells.

[0088] Experimental Example 2: Porosity Determination of Chlorella peptidonis

[0089] The lyophilized CP algae powder was used to determine the nitrogen adsorption isotherm and pore size distribution of the microalgae powder using a fully automated surface area and porosity analyzer (BET). Figure 3 As shown in Figures A and B, the nitrogen adsorption isotherm of *Chlorella proteoglycans* powder belongs to Type III isotherms. Type III isotherms typically appear on non-porous or macroporous solid materials, which may have relatively flat or rough surfaces but lack obvious pore structures. *Chlorella proteoglycans* powder has relatively few pores, with pore sizes mostly between 4 and 10 nm, and a few pores between 30 and 100 nm. Drug solutions can enter the interior of *Chlorella* through these larger pores.

[0090] Experimental Example 3: Micellar Particle Size Determination

[0091] Weigh 1.5 mL of the budesonide micelle solutions prepared in Examples 4, 5, 7, 9-13 respectively, filter them with a 2 μm filter, and then use a particle size analyzer to measure the particle size of the different micelle solutions. The results are shown in the table below.

[0092] Table 6: Particle size of budesonide micelle solutions prepared in Examples 4, 5, 7, 9-13

[0093]

[0094] As shown in Table 6, small molecule micelle materials tend to form smaller budesonide micelles, while the micelles formed by polymer materials have larger particle sizes and are difficult to enter the interior of Chlorella through pores.

[0095] Experimental Example 4: Determination of drug loading in microalgal drug compositions containing budesonide

[0096] Take 100 mg of each of the budesonide-containing microalgal drug compositions prepared in Examples 4, 5, 7, and 9, add them to 1 mL of pure water, adjust the pH to 4.5 with 2M HCl, add 100 mg of mannanase and 100 mg of pectinase, and enzymatically hydrolyze for 6 h at 45 ℃ and 100 rpm. Centrifuge at 5000 rpm for 4 min, discard the supernatant, add 1 mL of methanol to the precipitate, and extract by ultrasonication for 15 min. Centrifuge at 5000 rpm for 4 min, take the supernatant, dilute it 5 times with methanol, and determine the budesonide content in each example using high performance liquid chromatography. The specific conditions are shown in Table 7.

[0097] Table 7: Operating Conditions for High Performance Liquid Chromatography (UPLC)

[0098]

[0099] Table 8: Drug loading of budesonide-containing microalgal drug compositions prepared in Examples 4, 5, 7, and 9

[0100]

[0101] As shown in Table 8, budesonide has a higher drug loading when it is mixed with polyethylene glycol glycerol caprylate and caprylic acid capric acid in a ratio of 1:50.

[0102] Experimental Example 5: Fluorescence confocal imaging of microalgal drug compositions containing Nile Red dye

[0103] Take 100 mg of the microalgal drug composition containing Nile Red dye prepared in Examples 19-22 and dried Chlorella pyrenoidosa powder, disperse them in water, and observe the internal fluorescence of the microalgae using a laser confocal microscope.

[0104] like Figure 4As shown in Example 19, a significant amount of Nile Red fluorescence was observed inside the microalgae, indicating that when using a nonionic surfactant, a large number of hydrophobic model molecules, Nile Red, can enter the microalgae through passive water absorption and surface pores. When anionic, amphoteric, or cationic surfactants are used, fewer Nile Red molecules enter the microalgae due to the negative charge on the microalgae surface.

[0105] Experimental Example 6: Fluorescence imaging of microalgal drug compositions containing Nile Red dye in the gastrointestinal tract

[0106] Fifteen female ICR mice, weighing 20±2 g, were randomly divided into 10 groups. Each group was administered 0.2 mL of the microalgae drug composition containing Nile Red dye prepared in Example 19 and a Nile Red micelle solution (i.e., the Nile Red-caprylic / capric acid / polyethylene glycol glycerol micelle solution shown in the figure) via gavage. Mice were sacrificed at 1, 2, 4, 6, and 8 h after gavage, and the gastrointestinal tracts were harvested. The distribution of Nile Red dye and microalgae in the gastrointestinal tract of each group was observed using a small animal in vivo imaging system. The excitation wavelength of the Nile Red channel was 560 nm, and the emission wavelength was 640 nm; the excitation wavelength of the microalgae channel was 780 nm, and the emission wavelength was 820 nm.

[0107] like Figure 5 As shown, after oral gavage in Example 19, the distribution of Nile Red was lagging behind that of the microalgae at all sampling time points, indicating that the Nile Red dye molecules inside the microalgae can be continuously released after the microalgae-containing microalgae drug composition enters the gastrointestinal tract. Compared with the Nile Red-caprylic / capric acid / polyethylene glycol glycerol micelle solution, the oral gavage in Example 19 showed stronger fluorescence intensity, indicating that the microalgae acted as a "reservoir" for hydrophobic molecules, continuously releasing hydrophobic molecules into the intestine.

[0108] Ileum, cecum, and colon segments were collected separately. After washing, the segments were placed in 2 mL centrifuge tubes, and each segment was accurately weighed. Then, 1 mL of 80% methanol aqueous solution and 5 mm diameter grinding beads were added to the centrifuge tubes, and the mixture was ground in a grinder at 10,000 rpm for 5 min. The homogenized sample was centrifuged at 14,000 rpm for 5 min at 4 ℃, and the supernatant was collected. 200 μL of the supernatant was placed in a black 96-well plate, and the values ​​were read using a microplate reader. The distribution of NR Nile Red in each intestinal segment was then calculated.

[0109] Table 9: Concentrations (ng / g) of the Nile Red dye-containing microalgae drug composition and Nile Red-caprylate-capric acid-polyethylene glycol glycerol micelle solution prepared in Example 19 in the ileum, cecum, and colon.

[0110]

[0111] From Table 9 and Figure 6 As shown, quantitative analysis of the distribution of Nile Red dye in different intestinal segments revealed that after oral gavage for 4 h, 6 h, and 8 h, the Nile Red dye content in the ileum and cecum of mice in the group administered Nile Red-caprylic / capric acid / polyethylene glycol glycerol micelle solution was higher than that in the group administered Nile Red-caprylic / capric acid / polyethylene glycol glycerol micelle solution. This indicates that the present invention can promote the content and absorption of hydrophobic substances in the ileum, cecum, and other parts of the intestine.

[0112] Experiment Example 7: Investigation of the effects of thermosensitive gel and inorganic metal salt content on gel-forming factors

[0113] Take appropriate amounts of Soluplus® and sodium chloride, add 20 mL of pure water, stir at 4 ℃ for 4 h, then let stand at 4 ℃ for 12 hours to swell, thus preparing a gel solution. Separately weigh 20 mg of budesonide powder and dissolve it in 1 mL of octanoic acid-capric acid-polyethylene glycol glycerol ester. Slowly add 19 mL of the above gel solution, and stir vigorously at 4 ℃ and 1500 rpm for 2 hours to obtain a homogeneous gel solution. Investigate its gelation temperature and gelation behavior at 37 ℃.

[0114] Table 10: Test results of the effects of thermosensitive gel and inorganic metal salt content on gel-forming factors

[0115]

[0116] Note: " / " indicates good fluidity and cannot form a gel; "*" indicates a semi-transparent gel with a soft texture, which regains fluidity after being removed from 37°C and placed at room temperature for about 15 seconds; "**" indicates an opaque gel with a hard texture, which regains fluidity after being removed from 37°C and placed at room temperature for about 40 seconds; "***" indicates a milky white gel with a hard texture, which is difficult to regain fluidity after being removed from 37°C and placed at room temperature.

[0117] As shown in Table 10, the key performance parameters of the thermosensitive gel, such as gelation temperature and gel strength, are mainly dominated by the concentration of Soluplus®. The mechanism is that the Soluplus® concentration directly determines the hydrophobic interactions and entanglement density of the polymer chains, which is the basis for forming a three-dimensional network structure. In contrast, the concentration of inorganic salts modulates the hydrophilic-hydrophobic balance of the system to some extent through the ionic strength effect, thus having a secondary impact on performance. However, different types of metal ions (such as Na⁺, K⁺, and Ca²⁺) have similar electrical strength and hydration capacity, and their effects on gel performance are not significantly different.

[0118] Experimental Example 8: In vitro release experiment of microalgal drug composition containing budesonide

[0119] Budesonide suspension (containing 1 mg of budesonide), the budesonide-containing microalgae drug composition prepared in Example 4, and the budesonide gel-containing microalgae drug composition prepared in Example 23 were placed in 50 mL centrifuge tubes, preheated at 37 °C for 1 h, and then 5 mL of simulated gastric fluid (pH 1.8) containing 1% Tween 80 was added. After stirring continuously at 37 °C for 2 h, 15 mL of simulated intestinal fluid (pH 7.4) containing 1% Tween 80 was added. At 0.5, 1, 2, 3, 4, 6, 8, and 12 h after adding the simulated gastric fluid, a certain volume of release medium was taken, centrifuged at 14000 rpm, and the supernatant was collected. The drug concentration at different time points was determined by high performance liquid chromatography, and the drug release rate was calculated. At the same time, an equal volume of release medium was added.

[0120] Table 11: In vitro release rate (%) of budesonide-containing compositions

[0121]

[0122] From Table 11 and Figure 7 It was observed that the budesonide suspension was completely released in simulated digestive fluid. In contrast, the release rates of Examples 4 and 23 in simulated gastric fluid were both low (less than 10%) within 2 hours, indicating that the microalgae carrier has a protective effect on budesonide in the stomach. In the subsequent 2–12 hours in simulated intestinal fluid, Example 23 exhibited a slower release rate compared to Example 4, indicating that the added Soluplus® has a sustained-release effect, which helps the microalgae drug composition achieve a longer-lasting targeted release in the intestine.

[0123] Example 9: Evaluation of in vivo pharmacokinetics and tissue distribution of microalgal drug compositions containing budesonide

[0124] Forty-eight healthy male Wistar rats (weighing 180–220 g) were acclimatized for one week and then fasted for 12 hours before being randomly divided into three groups (n=16 / group). The rats were administered budesonide suspension orally via gavage, as in Example 4 and Example 23, respectively, at a dose of 2 mg / kg. Blood samples were collected from the orbital sinus at 1, 2, 4, and 8 hours after administration (n=4 at each time point). After euthanasia, the rats were dissected, and tissues from the stomach, ileum, cecum, and colon were collected. All gastrointestinal tissues were rinsed with PBS buffer to remove contents, dried, and weighed.

[0125] The plasma sample processing method is as follows: take 100 μL of plasma, add 500 μL of methanol, vortex mix for 5 min, centrifuge at 12000 rpm and 4 ℃ for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane for testing;

[0126] The tissue sample processing method is as follows: accurately weigh a quantitative amount of tissue, add 1 mL of methanol, homogenize in a low-temperature grinder (100 Hz, 4℃) for 10 min, centrifuge at 5000 rpm, 4℃ for 10 min, take 100 μL of the supernatant, add 500 μL of methanol, vortex mix for 5 min, centrifuge at 12000 rpm, 4℃ for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane before analysis.

[0127] Budesonide concentrations in plasma and tissue samples were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The chromatographic and mass spectrometric conditions are shown in the table below.

[0128] Table 12: Conditions for UPLC-MS / MS

[0129]

[0130] Table 13: Budesonide drug concentrations in rat plasma, gastric tissue, and parts of the intestine at different time points

[0131]

[0132] From Table 13 and Figure 8 It was observed that the plasma budesonide concentration remained at a low level at all time points. After 1 hour, there was no significant difference in plasma budesonide levels among the budesonide suspension group, Example 4 group, and Example 23 group. This result indicates that the amount of budesonide entering the bloodstream is relatively small, resulting in low systemic exposure. The concentration of budesonide in gastric tissue showed that the budesonide suspension group > Example 4 group > Example 23 group, indicating that the microalgae carrier can reduce budesonide release in the stomach. The distribution of budesonide content in the ileum, cecum, and colon was: Example 23 group > Example 4 group > budesonide suspension group. These results demonstrate that the microalgae-based budesonide drug composition enhances the release of budesonide in the ileum, cecum, and colon.

[0133] Experimental Example 10: Evaluation of the therapeutic effect of a microalgae drug composition containing budesonide on mice with dextran sulfate-induced colitis.

[0134] (1) Experimental animals: 32 healthy male C57BL / 6 mice with a weight range of 18-22 g were selected. They were acclimatized for 3 days under standard conditions before the experiment.

[0135] (2) Preliminary group modeling and model verification:

[0136] After acclimatization, the mice were randomly divided into two groups:

[0137] ① Healthy control group (Control, n=7): Free access to sterile water for 7 days;

[0138] ②DSS Model Group (n=25): Individuals were given free access to 2.5% (w / v) sodium dextran sulfate (DSS) aqueous solution for 7 days to induce acute colitis.

[0139] After the modeling period ended (day 7), one mouse was randomly selected from the healthy control group and the DSS model group. After being fully anesthetized with isoflurane, the mice were euthanized by cervical dislocation. Colon tissue was quickly harvested, fixed in 4% paraformaldehyde solution, and then embedded in paraffin, sectioned (4 μm thick), and stained with hematoxylin and eosin (H&E). Histopathological examination (observing typical pathological features, such as crypt structure destruction and inflammatory cell infiltration) confirmed the successful establishment of colitis model in the DSS model group mice.

[0140] (3) Treatment group:

[0141] After confirming the model's success, the remaining DSS model mice (n=24) were re-randomized into 4 groups (n=6 / group), which, together with the healthy group, were used for subsequent treatment evaluation. The grouping and experimental procedures are as follows:

[0142] ① Healthy group: Administered an equal volume of physiological saline via gavage daily;

[0143] ② Model group: Mice in the DSS model group were administered an equal volume of physiological saline by gavage daily;

[0144] ③Budesonide suspension group: DSS model mice were given budesonide suspension by gavage daily, 3 mg / kg / d;

[0145] ④ Example 4 group: DSS model mice were administered Example 4 via gavage daily at a dose of 3 mg / kg / d;

[0146] ⑤ Example 23 group: DSS model mice were administered Example 23 by gavage daily at a dose of 3 mg / kg / d.

[0147] (4) Evaluation indicators and methods

[0148] ① General condition monitoring: Record daily changes in weight, stool characteristics (Bristol classification), and survival status;

[0149] ②Dissection and sampling: Samples were collected under isoflurane anesthesia 24 hours after the last administration.

[0150] Blood: Blood was collected from the orbital venous plexus and the serum was separated by centrifugation (stored at -80 ℃).

[0151] Spleen: Calculate the spleen index by weighing (spleen weight mg / body weight g × 100%).

[0152] Colon: Measure the length and weigh it, and calculate the colon weight / length ratio (mg / cm).

[0153] (5) Histological analysis:

[0154] Proximal colon segments were harvested, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned (4 μm); H&E staining was performed, and histopathological evaluation was conducted according to the Dieleman scoring system (0-4 points).

[0155] Table 14: Evaluation of the therapeutic effects of budesonide-containing microalgae drug compositions on DSS model mice

[0156]

[0157] From Table 14 and Figure 9 It was found that the colon length of mice in the DSS model group was significantly shorter than that of mice in the healthy group. Mice treated with budesonide suspension, oral gavage (Examples 4 and 23) all had significantly longer colons than the DSS model group (p<0.05), with the treatment group in Example 23 showing significantly better results than the budesonide group (p<0.05). Figure 9 (A, C in the text). The DAI score results showed that Example 23 was more effective in improving disease activity, and its therapeutic effect was significantly better than the budesonide treatment group and the Example 4 treatment group ( Figure 9 (B) In the DSS model group, the colon thickened and its wet weight increased due to inflammatory cell infiltration and tissue proliferation, resulting in a significantly elevated colon weight-to-length ratio; in contrast, the treatment group in Example 23 effectively reduced this ratio and alleviated colonic swelling symptoms. Figure 9 The spleen coefficient (spleen weight / body weight × 10) in the DSS model group mice was significantly higher than that in the healthy group, indicating a stronger systemic immune response; while the spleen coefficient was significantly reduced in all groups treated with budesonide suspension, oral gavage (Examples 4 and 23). Figure 9 E in the text). Histopathological analysis showed that the colon structure of healthy mice was intact, with no obvious inflammatory lesions; the colon tissue of mice in the DSS model group was severely damaged, with significant inflammatory cell infiltration; the budesonide suspension group did not significantly improve colon inflammation; while the degree of colon tissue damage and the level of inflammatory cell infiltration in the treatment group of Example 23 were significantly reduced (E in the text). Figure 9 (F and G in the text).

[0158] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microalgae drug composition for targeted and controlled release into the gut, characterized in that, The invention comprises a hydrophobic drug, a micelle material, and microalgae, wherein the hydrophobic drug is loaded onto the micelle material to form drug-loaded micelles, and the drug-loaded micelles are located inside the microalgae. The hydrophobic drug is budesonide; the micelle material is polyethylene glycol glycerol caprylate-capric acid, and the mass ratio of the hydrophobic drug to the micelle material is 1:40~60. The microalgae mentioned are one or more of Spirulina and Chlorella; The mass ratio of the hydrophobic drug to the microalgae is 1:50~1000.

2. The intestinal-targeted controlled-release microalgae drug composition according to claim 1, characterized in that, The microalgae mentioned is Chlorella vulgaris.

3. The intestinal-targeted controlled-release microalgae drug composition according to claim 1, characterized in that, The particle size of the drug-loaded micelles is 10~1200 nm.

4. The intestinal-targeted controlled-release microalgae drug composition according to claim 1, characterized in that, The intestinal-targeted controlled-release microalgae drug composition also contains a gel material and a metal inorganic salt. The gel material is a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and the inorganic salt is one of sodium chloride, potassium chloride, and calcium chloride. The mass ratio of the hydrophobic drug to the gel material is 1:190, 1:237.5, 1:285, or 1:332.

5. When the mass ratio of hydrophobic drug to gel material is 1:190, the mass ratio of hydrophobic drug to metal inorganic salt is 1:19~47.

5. When the mass ratio of the hydrophobic drug to the gel material is 1:237.5, the mass ratio of the hydrophobic drug to the metal inorganic salt is 1:8.55~47.

5. When the mass ratio of the hydrophobic drug to the gel material is 1:285, the mass ratio of the hydrophobic drug to the metal inorganic salt is 1:0.95~19.

5. The use of the intestinal-targeted controlled-release microalgae drug composition according to any one of claims 1 to 4 in the preparation of a drug for treating inflammatory bowel disease.

Images