A kind of astaxanthin-loaded nanoparticle and its preparation method and application

Abstract

The application relates to the technical field of medicines, and discloses a kind of astaxanthin-loaded nanoparticles and its preparation method and application.The nanoparticles take astaxanthin / mucin complex formed by astaxanthin and mucin as inner core, and the inner core surface is coated with polydopamine shell layer, forming astaxanthin / mucin@polydopamine nanoparticles with core-shell structure.The application utilizes the complex of mucin and astaxanthin, and the polydopamine shell layer generated in situ on the surface of the complex, significantly improves the stability of astaxanthin in gastric juice environment, and the particle size is maintained at about 250 nm without significant aggregation;At the same time, the obtained nanoparticles have significant antioxidant effect, and can affect the fibrosis microenvironment to realize targeted slow release.In vitro experiments show that the nanoparticles can effectively inhibit the expression of macrophage inflammatory factor IL-1beta, and significantly reduce the level of alpha-SMA in intestinal fibroblasts, thereby blocking the intestinal fibrosis process.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, to a drug delivery system for astaxanthin, and particularly to astaxanthin-loaded nanoparticles, their preparation methods, and applications. Background Technology

[0002] Crohn's disease (CD) is a chronic inflammatory bowel disease. More than half of patients develop secondary intestinal fibrosis, leading to intestinal stenosis and obstruction, ultimately requiring surgical resection of the obstructed bowel segment to relieve symptoms. Clinical data shows that the obstruction rate after bowel segment resection in CD patients exceeds 50%, necessitating multiple surgeries, severely impacting quality of life and even endangering life. Currently, there are no effective drugs for treating intestinal fibrosis. Existing anti-inflammatory drugs and biologics can improve the control of intestinal inflammation, but they are ineffective in controlling the progression of fibrosis. Therefore, there is an urgent need to develop highly effective and precise drugs specifically targeting intestinal fibrosis in CD to block the fibrotic process.

[0003] Under CD pathological conditions, initial inflammatory biochemical signals stimulate abnormal activation of key effector cells, intestinal myofibroblasts, leading to excessive secretion of extracellular matrix (ECM). Subsequently, increased ECM deposition stiffness results in abnormal mechanical and energy signals, continuously stimulating fibroblast activation and further promoting ECM secretion, thus forming a positive feedback loop independent of the initial inflammatory stimulus. Mitochondria, as intracellular signal transduction centers, integrate various biochemical, mechanical, and metabolic signals. In the fibrotic microenvironment, mitochondrial dysfunction leads to a state of persistent oxidative stress, triggering excessive production of reactive oxygen species (ROS) and amplifying pathological signals. Therefore, treatment targeting mitochondrial dysfunction and oxidative stress holds promise for breaking this vicious cycle.

[0004] Astaxanthin (AST) is a natural carotenoid with powerful antioxidant capabilities. It can scavenge reactive oxygen species (ROS) in mitochondria and reduce oxidative stress, making it a highly efficient mitochondrial-targeted antioxidant. However, its clinical translation is limited due to its low bioavailability and extreme instability in the gastrointestinal environment. Existing technologies, such as Chinese Patent Application Publication No. CN121930383A, disclose a folic acid-modified reactive oxygen species-responsive sodium alginate nanocarrier and its application. This nanocarrier introduces a double selenium bond ROS-responsive unit and a folic acid-targeting molecule to encapsulate and protect astaxanthin. Stable in the gastrointestinal environment, upon reaching the site of colonic inflammation, the nanocarrier can respond to high ROS levels and achieve intelligent sustained drug release through folic acid receptor targeting. However, its preparation involves multiple raw materials, making the preparation process complex and difficult to operate. Therefore, more drug delivery systems targeting AST are urgently needed.

[0005] Developing a structurally stable AST delivery system with targeted release capability and high bioavailability to fully realize its therapeutic potential in targeting and regulating mitochondrial homeostasis and combating intestinal fibrosis is key to the clinical translation of AST. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides nanoparticles loaded with astaxanthin, their preparation method, and their applications.

[0007] The specific technical solution of this invention is as follows:

[0008] In a first aspect, the present invention provides nanoparticles loaded with astaxanthin. The nanoparticles have a core-shell structure, wherein the core of the core-shell structure is an astaxanthin / mucin complex formed by astaxanthin and mucin, and the shell layer of the core-shell structure is polydopamine.

[0009] This invention uses mucin as a protective carrier and utilizes a polydopamine shell generated in situ on the surface of the complex to significantly improve the stability of astaxanthin in the gastric juice environment, maintaining the particle size at around 250 nm without aggregation; it achieves improved AST bioavailability and stable AST structure, enabling it to be effectively applied to anti-intestinal fibrosis.

[0010] Furthermore, after incubation in simulated gastric juice for 1 hour, the nanoparticles showed a particle size change of less than 200 nm. The highly stable nanoparticles of this invention exhibit superior anti-inflammatory and anti-intestinal fibrosis effects.

[0011] Secondly, the present invention provides a method for preparing astaxanthin-loaded nanoparticles, characterized by comprising the following steps:

[0012] (1) Mix astaxanthin with mucin to prepare a mixture of astaxanthin and mucin, sonicate and rotary evaporate to obtain AST / Muc complex;

[0013] (2) Dissolve dopamine in buffer solution and then add it to the AST / Muc complex obtained in step (1). React in the dark to form a polydopamine shell attached to the surface of the astaxanthin / mucin complex, and obtain AST / Muc@PDA nanoparticles.

[0014] Furthermore, in step (1), the mass ratio of astaxanthin to mucin is 1:5~15.

[0015] Furthermore, in step (1), the ultrasonic treatment time is 2 to 10 minutes.

[0016] Further, in step (1), the method for mixing astaxanthin and mucin is as follows: dissolve astaxanthin in an organic solvent to prepare an astaxanthin solution; dissolve mucin in a buffer solution to prepare a mucin solution; mix the astaxanthin solution and the mucin solution to obtain a mixture of astaxanthin and mucin.

[0017] The organic solvent is selected from one or more of dichloromethane, ethanol, methanol, and tetrahydrofuran, and the pH of the buffer solution is 7.0 to 9.0.

[0018] Furthermore, in step (2), the mass ratio of astaxanthin to dopamine is 1:1~10.

[0019] Furthermore, in step (2), the temperature for the light-protected reaction is 30~42℃, and the reaction time is 7~10 hours.

[0020] Furthermore, in step (2), the light-avoiding reaction is carried out under the conditions of shaking and / or stirring on a shaker, and the stirring speed is 80~200 rpm.

[0021] Thirdly, this invention provides the application of astaxanthin-loaded nanoparticles in the preparation of drugs for treating intestinal fibrosis. In vitro experiments show that the above-mentioned nanoparticles can effectively inhibit the expression of the macrophage inflammatory factor IL-1β and significantly reduce the level of α-SMA in intestinal fibroblasts, thereby blocking the process of intestinal fibrosis.

[0022] Compared with the prior art, the present invention has the following technical effects:

[0023] (1) This invention provides astaxanthin / mucin@polydopamine nanoparticles with a core-shell structure. The nanoparticles have an astaxanthin / mucin complex formed by astaxanthin and mucin as the core, and the core surface is coated with a polydopamine shell. This invention uses mucin as a protective carrier and utilizes the polydopamine shell generated in situ on the surface of the complex to significantly improve the stability of astaxanthin in the gastric juice environment, maintain the particle size at around 250 nm and avoid aggregation; it achieves improved AST bioavailability and stable AST structure, enabling it to be effectively applied to anti-intestinal fibrosis. In vitro experiments show that the nanoparticles can effectively inhibit the expression of macrophage inflammatory factor IL-1β and significantly reduce the level of α-SMA in intestinal fibroblasts, thereby blocking the process of intestinal fibrosis, and have the potential to be used to prepare oral drugs for the prevention or treatment of intestinal fibrosis.

[0024] (2) This invention uses mucin as a protective carrier and utilizes a polydopamine shell generated in situ on the surface of the complex. Through the synergistic effect of astaxanthin, polydopamine, and mucin, specifically, mucin disperses and protects astaxanthin, improving its stability; polydopamine, as the shell, not only provides shell protection for astaxanthin but also utilizes its own ability to scavenge reactive oxygen species. Through these multiple effects, it exerts a synergistic antioxidant effect with astaxanthin, resulting in a significant increase in DPPH scavenging rate.

[0025] (3) The preparation method of the core-shell structured astaxanthin / mucin@polydopamine nanoparticles of the present invention is simple, involving only 3 reaction raw materials and simple stirring reaction. Attached Figure Description

[0026] Figure 1 The results show the in vitro particle size variation of AST / Muc@PDA.

[0027] Figure 2 The results show the in vitro scavenging capacity of AST / Muc@PDA against reactive oxygen species.

[0028] Figure 3 The results show the in vitro cell anti-inflammatory activity of AST / Muc@PDA.

[0029] Figure 4 The results show the in vitro antifibrotic ability of AST / Muc@PDA. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0031] In the following examples, the mucin used was purchased from Beijing Puxitang Biotechnology Co., Ltd., CAS No: 84082-64-4.

[0032] Example 1

[0033] An oral nanoparticle for anti-intestinal fibrosis, AST / Muc@PDA, is prepared according to the following steps:

[0034] Step S1: Dissolve 10 mg of mucin in 2 mL of 0.01 M Tris-HCl buffer (pH 8.5) and sonicate until completely dissolved to obtain a mucin solution. Separately, dissolve 1 mg of AST in 500 μL of dichloromethane (DCM) to obtain an AST solution. Mix the AST solution and the mucin solution at a ratio of 1:4 (v / v), sonicate for 2 minutes, and then completely remove the organic solvent using a rotary evaporator to obtain the AST / Muc complex.

[0035] Step S2: Weigh 6 mg of dopamine and dissolve it in 2 mL of 0.01 M Tris-HCl solution (pH=8.5), then add it to the AST / Muc composite prepared above. The resulting mixture is then incubated in the dark at 37°C and 100 rpm for 8 hours. Dopamine undergoes a self-polymerization reaction on the surface of the AST / Muc composite to form a PDA shell, thus obtaining AST / Muc@PDA nanoparticles.

[0036] Step S3: The obtained AST / Muc@PDA nanoparticles were tested and found to have a particle size of 243 nm. After incubation with simulated gastric juice (SGF) containing pepsin for 2 hours, the particle size change was less than 50 nm. This shows that the particle size of the AST / Muc@PDA nanoparticles remained within a stable nanoscale range.

[0037] Example 2

[0038] An oral nanoparticle for anti-intestinal fibrosis, AST / Muc@PDA, is prepared according to the following steps:

[0039] Step S1: Dissolve 5 mg of mucin in 2 mL of 0.01 M Tris-HCl buffer (pH 8.5) and sonicate until completely dissolved to obtain a mucin solution. Separately, dissolve 1 mg of AST in 500 μL of DCM to obtain an AST solution. Mix the AST solution and the mucin solution at a ratio of 1:4 (v / v), sonicate for 2 minutes, and then use a rotary evaporator to completely remove the organic solvent to obtain the AST / Muc complex.

[0040] Step S2: Weigh 3 mg of dopamine and dissolve it in 2 mL of 0.01 M Tris-HCl solution (pH=8.5), then add it to the AST / Muc composite prepared above. The resulting mixture is then incubated in the dark at 37°C and 100 rpm for 9 hours. Dopamine undergoes a self-polymerization reaction on the surface of the AST / Muc composite to form a PDA shell, thus obtaining AST / Muc@PDA nanoparticles.

[0041] Step S3: The obtained AST / Muc@PDA nanoparticles were tested and found to have a particle size of 246 nm. After incubation with simulated gastric juice (SGF) containing pepsin for 2 hours, the particle size change was less than 50 nm. This shows that the particle size of the AST / Muc@PDA nanoparticles remained within a stable nanoscale range.

[0042] Example 3

[0043] An oral nanoparticle for anti-intestinal fibrosis, AST / Muc@PDA, is prepared according to the following steps:

[0044] Step S1: Dissolve 15 mg of mucin in 2 mL of 0.01 M Tris-HCl buffer (pH 8.5) and sonicate until completely dissolved to obtain a mucin solution. Separately, dissolve 1 mg of AST in 500 μL of DCM to obtain an AST solution. Mix the AST solution and the mucin solution at a ratio of 1:4 (v / v), sonicate for 2 minutes, and then completely remove the organic solvent using a rotary evaporator to obtain the AST / Muc complex.

[0045] Step S2: Weigh 10 mg of dopamine and dissolve it in 2 mL of 0.01 M Tris-HCl solution (pH=8.5), then add it to the AST / Muc composite prepared above. The resulting mixture is then incubated in the dark at 37°C and 100 rpm for 10 hours. Dopamine undergoes a self-polymerization reaction on the surface of the AST / Muc composite to form a PDA shell, thus obtaining AST / Muc@PDA nanoparticles.

[0046] Step S3: The obtained AST / Muc@PDA nanoparticles were tested and found to have a particle size of 254 nm. After incubation with simulated gastric juice (SGF) containing pepsin for 2 hours, the particle size change was less than 40 nm. This shows that the particle size of the AST / Muc@PDA nanoparticles remained within a stable nanoscale range.

[0047] Performance Characterization

[0048] The AST / Muc@PDA nanoparticles obtained in Example 1 were tested for the following properties.

[0049] (1) Gastric juice stability test

[0050] The prepared AST / Muc and AST / Muc@PDA were incubated in simulated gastric juice (SGF) supplemented with pepsin, respectively. The particle size changes of the two formulations after incubation in SGF for 0, 1, and 2 hours were investigated. The results are shown in [Figure number missing]. Figure 1 The particle size change over 2 hours is the particle size measured at 2 hours minus the particle size measured at 0 hours.

[0051] Depend on Figure 1 It was found that both particles maintained a stable particle size of around 250 nm in the initial state; however, after incubation in SGF, the particle size of the AST / Muc complex without PDA encapsulation increased, indicating that its structure underwent disintegration or aggregation. In contrast, the particle size change of AST / Muc@PDA was relatively small, with a change of less than 50 nm after 2 hours, maintaining a more stable nanoscale size range. This suggests that the rigid PDA shell significantly improved the structural integrity of the formulation in gastric juice.

[0052] (2) In vitro assessment of reactive oxygen species scavenging capacity

[0053] The in vitro antioxidant capacity of the formulations was evaluated using a DPPH free radical scavenging assay. The AST / Muc complex and AST / Muc@PDA were mixed with DPPH, incubated in the dark for 30 minutes, and their free radical scavenging capacity was measured spectrophotometrically. Results are shown below. Figure 2 .

[0054] Depend on Figure 2 The experimental results show that the DPPH scavenging rate of the AST / Muc complex simply encapsulated in mucin is low. However, the scavenging rate of the AST / Muc@PDA nanoformulation significantly increased after adding a PDA shell. This result fully demonstrates that the PDA shell plays a significant synergistic role with the core drug AST in terms of antioxidant properties.

[0055] (3) In vitro anti-inflammatory capacity assessment

[0056] In in vitro immunomodulatory experiments, RAW 264.7 macrophages were seeded in 12-well culture plates. The control group received physiological saline, while the model group received lipopolysaccharide (LPS) stimulation for 24 hours to induce an inflammatory response (denoted as TGF-β). The drug-treated groups received AST monotherapy, blank vector Muc@PDA nanoparticles, and AST / Muc@PDA nanoparticles, respectively. The inhibition of pro-inflammatory cytokine IL-1β expression in different drug-treated groups was detected by qRT-PCR. Results are shown below. Figure 3 .

[0057] like Figure 3 As shown, the relative expression level of IL-1β in the normal control group was extremely low, while the expression level in the LPS-stimulated group was significantly increased. After treatment with free astaxanthin, blank carrier Muc@PDA, and the AST / Muc@PDA nanoformulation of this invention, the relative expression level of intracellular IL-1β was significantly inhibited in all groups. Furthermore, compared to other treatment groups, AST / Muc@PDA showed the best anti-inflammatory level. This confirms the excellent efficacy of this nanodelivery system in blocking inflammatory signal transduction and alleviating oxidative stress-induced intestinal inflammation.

[0058] Furthermore, in in vitro immunomodulatory experiments, RAW 264.7 macrophages were seeded in 12-well culture plates. The control group received physiological saline, while the model group was stimulated with lipopolysaccharide (LPS) for 24 hours to induce an inflammatory response (denoted as TGF-β). The treatment groups received AST / Muc complex and AST / Muc@PDA nanoparticles, respectively. The inhibition of pro-inflammatory cytokine IL-1β expression in different treatment groups was detected by qRT-PCR. The results showed that the relative expression level of IL-1β in the control group was extremely low, while the expression level in the LPS-stimulated group was significantly increased. After the addition of AST / Muc complex and AST / Muc@PDA nanoparticles, the relative expression level of intracellular IL-1β was inhibited to varying degrees, with the inhibitory effect being stronger after the addition of AST / Muc@PDA nanoparticles. This indicates that the more stable AST / Muc@PDA nanoparticles can exert a sustained anti-inflammatory effect, thus exhibiting a stronger inhibitory effect on IL-1β expression.

[0059] (4) In vitro anti-fibrotic ability assessment

[0060] Intestinal fibroblasts were harvested and seeded into 6-well plates. The control group received physiological saline, the model group was pretreated with TGF-β1, and the drug-treated groups received AST monotherapy, blank vector Muc@PDA nanoparticles, and AST / Muc@PDA nanoparticles, respectively. The mRNA levels of α-SMA in different drug-treated groups were detected by qRT-PCR. Results are shown below. Figure 4 .

[0061] Depend on Figure 4 It was found that the expression level of α-SMA was significantly upregulated in the TGF-β-induced group compared with the control group. After intervention with free AST, Muc@PDA, and AST / Muc@PDA nanoparticles, the expression level of α-SMA decreased significantly. In particular, the AST / Muc@PDA group showed the lowest α-SMA expression, fully demonstrating that the AST / Muc@PDA nanoparticles can effectively inhibit the activation of myofibroblasts and possess great therapeutic potential in blocking the progression of intestinal fibrosis.

[0062] Furthermore, intestinal fibroblasts were harvested and seeded into 6-well plates. The control group received physiological saline, the model group was pretreated with TGF-β1, and the drug-treated groups received AST / Muc complex and AST / Muc@PDA nanoparticles, respectively. qRT-PCR was used to detect α-SMA mRNA levels in different drug-treated groups. The results showed that α-SMA expression was relatively low in the control group, while it was significantly increased in the LPS-stimulated group. After the addition of AST / Muc complex and AST / Muc@PDA nanoparticles, α-SMA expression levels decreased significantly to varying degrees, with the addition of AST / Muc@PDA nanoparticles resulting in even lower α-SMA expression levels. This indicates that the more stable AST / Muc@PDA nanoparticles can exert a sustained anti-inflammatory effect and provide sustained drug release, thus exhibiting a stronger inhibitory effect on myofibroblast activation.

[0063] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A nanoparticle loaded with astaxanthin, characterized in that: The nanoparticles have a core-shell structure, with the core being an astaxanthin / mucin complex formed by astaxanthin and mucin, and the shell being polydopamine.

2. The astaxanthin-loaded nanoparticles as described in claim 1, characterized in that: After incubation in simulated gastric juice for 1 hour, the nanoparticles showed a particle size change of less than 200 nm.

3. A method for preparing astaxanthin-loaded nanoparticles, characterized in that: Includes the following steps: (1) Mix astaxanthin with mucin to prepare a mixture of astaxanthin and mucin, sonicate and rotary evaporate to obtain AST / Muc complex; (2) Dissolve dopamine in buffer solution and then add it to the AST / Muc complex obtained in step (1). React in the dark to form a polydopamine shell attached to the surface of the astaxanthin / mucin complex, and obtain AST / Muc@PDA nanoparticles.

4. The preparation method according to claim 3, characterized in that: In step (1), the mass ratio of astaxanthin to mucin is 1:5~15.

5. The preparation method according to claim 3, characterized in that: In step (1), the ultrasonic treatment time is 2 to 10 minutes.

6. The preparation method according to claim 3, characterized in that: In step (1), the method for mixing astaxanthin and mucin is as follows: dissolve astaxanthin in an organic solvent to prepare an astaxanthin solution; dissolve mucin in a buffer solution to prepare a mucin solution; mix the astaxanthin solution and the mucin solution to obtain a mixture of astaxanthin and mucin. The organic solvent is selected from one or more of dichloromethane, ethanol, methanol, and tetrahydrofuran, and the pH of the buffer solution is 7.0 to 9.

0.

7. The preparation method according to claim 3, characterized in that: In step (2), the mass ratio of astaxanthin to dopamine is 1:1~10.

8. The preparation method according to claim 3, characterized in that: In step (2), the temperature for the reaction in the dark is 30~42℃ and the reaction time is 7~10 hours.

9. The preparation method according to claim 3, characterized in that: In step (2), the light-avoiding reaction is carried out under the conditions of shaking and / or stirring on a shaker, and the stirring speed is 80~200 rpm.

10. The use of the astaxanthin-loaded nanoparticles as described in any one of claims 1 to 2 or the nanoparticles prepared by the preparation method as described in any one of claims 3 to 9 in the preparation of drugs for anti-intestinal fibrosis.

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