A pH-responsive fish gelatin-chondroitin sulfate nanoparticle for loading fucoxanthin and its preparation method

Abstract

This invention provides pH-responsive fish gelatin-chondroitin sulfate nanoparticles, prepared from fish gelatin and chondroitin sulfate in a mass ratio of 1-3 parts fish gelatin and 0.5-1.5 parts chondroitin sulfate. The invention also provides a method for preparing these pH-responsive fish gelatin-chondroitin sulfate nanoparticles and their applications. Furthermore, this invention provides pH-responsive fish gelatin-chondroitin sulfate nanoparticles for loading fucoxanthin. To achieve stable gastric juice and rapid intestinal juice release of fucoxanthin, this invention introduces a biocompatible anionic polysaccharide—chondroitin sulfate—into fish gelatin. pH-responsive nanoparticles are prepared using a simple composite coagulation method, enabling fucoxanthin to achieve stable gastric juice and rapid intestinal juice release, thus improving the stability and bioavailability of fucoxanthin. This provides a new option for the research of fucoxanthin stabilization and delivery systems. In addition, this invention expands the application of chondroitin sulfate in the food industry.

Description

Technical Field

[0001] This invention relates to a pH-responsive fish gelatin-chondroitin sulfate nanoparticle for loading fucoxanthin and its preparation method, belonging to the field of pharmaceutical preparations. Background Technology

[0002] Fish gelatin is a natural material extracted from fish skin, bones, and scales. Compared to mammalian gelatin, it has advantages such as lower production cost, lower immunogenicity, and fewer religious restrictions. Methacrylated fish gelatin was synthesized by grafting photocrosslinking groups onto fish gelatin. Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) composed of alternating sugar chains (N-acetylgalactosamine and glucuronic acid). It typically attaches to proteins as part of a proteoglycan. Chondroitin chains can contain over 100 individual sugars, each of which can be sulfated at different positions and in greater quantities. Chondroitin sulfate is an important structural component of cartilage and provides most of its compressibility. Along with glucosamine, chondroitin sulfate has become a widely used dietary supplement for the treatment of osteoarthritis.

[0003] Currently, there are no literature reports on the preparation of nanoparticles from gelatin and chondroitin sulfate composites, or their use as drug carriers.

[0004] Fucoxanthin is the most abundant carotenoid in nature. It possesses excellent biological activities, including reducing oxidative stress, decreasing inflammation, combating obesity, and fighting cancer, making it widely applicable in human health. However, due to its unique functional groups, fucoxanthin is easily degraded by heat, light, and oxygen, and has poor water solubility, limiting its application in the food industry. To address the technical issues of fucoxanthin's instability and poor water solubility, some literature reports on its preparation as microcapsules. Application number: 201610059392.8, invention title: A method for preparing fucoxanthin microcapsules, discloses a method for preparing fucoxanthin microcapsules, belonging to the field of food processing technology. The specific steps are: (1) After uniformly mixing palm stearin and fucoxanthin, use it as the core material and sonicate it in a gelatin solution to form a uniform emulsion; (2) Add a gum arabic solution containing surfactant to the emulsion described in step (1), stir at a constant temperature for 5-15 minutes, adjust the pH to 3.8-4.2 with acetic acid, and carry out a coagulation reaction; (3) Cool the system described in step (2) to below 10°C, add tannic acid solution to solidify the microcapsules; (4) After the microcapsules precipitate, wash with deionized water, repeat several times, and freeze-dry to obtain microcapsule powder. This application involves many excipients and has a complex preparation process, making it unsuitable for large-scale production.

[0005] Nanoencapsulation can protect unstable compounds from adverse conditions during processing, storage, and transportation, thereby improving their stability and bioavailability. Furthermore, nanoencapsulation may also enable the controlled release of bioactive compounds. Summary of the Invention

[0006] The technical solution of the present invention is to provide a pH-responsive fish gelatin-chondroitin sulfate nanoparticle, and another technical solution of the present invention is to provide a pH-responsive fish gelatin-chondroitin sulfate nanoparticle for loading fucoxanthin.

[0007] This invention provides pH-responsive fish gelatin-chondroitin sulfate nanoparticles, which are prepared from fish gelatin and chondroitin sulfate in a mass ratio of:

[0008] Fish gelatin 1-3 parts, chondroitin sulfate 0.5-1.5 parts.

[0009] More preferably, it is prepared from fish gelatin and chondroitin sulfate in a mass ratio of:

[0010] Two parts fish gelatin and one part chondroitin sulfate.

[0011] The present invention also provides a method for preparing the pH-responsive fish gelatin-chondroitin sulfate nanoparticles, which includes the following steps:

[0012] a. Weigh out the raw materials fish gelatin and chondroitin sulfate in the specified weight ratios;

[0013] b. Preparation of fish gelatin stock solution: Dissolve freeze-dried fish gelatin in deionized water, and stir magnetically at 40-50℃ and 450-550rpm for 1-2h. After cooling to room temperature, adjust the pH value of the nanoparticles to 2.0-10.0. Then filter with a 0.45μm membrane to remove insoluble solids to obtain fish gelatin stock solutions of different concentrations, with concentrations of 1-10mg / mL.

[0014] c. Preparation of chondroitin sulfate stock solution: Chondroitin sulfate was dissolved in deionized water and magnetically stirred at 450-550 rpm for 1-2 hours at room temperature. The pH of the nanoparticles was adjusted to 2.0-10.0. Then, the solution was filtered through a 0.45 μm membrane to obtain chondroitin sulfate stock solutions of different concentrations, with concentrations of 1-10 mg / mL.

[0015] d. Preparation of fish gelatin-chondroitin sulfate nanoparticles:

[0016] The chondroitin sulfate stock solution prepared in step c was added to the fish gelatin stock solution prepared in step b at a volume ratio of 1:1. The mixture was stirred for 30 minutes at room temperature using a magnetic stirrer (500 rpm) to obtain gelatin chondroitin sulfate nanoparticles with different mass ratios.

[0017] More preferably, the preparation method of the gelatin stock solution in step b is as follows: lyophilized fish gelatin is dissolved in deionized water, magnetically stirred at 45°C and 500 rpm for 1 hour, cooled to room temperature, and then adjusted to pH 4 for the nanoparticles. Insoluble solids are then removed by filtration through a 0.45 μm membrane to obtain the fish gelatin stock solution. The preparation method of the chondroitin sulfate stock solution in step c is as follows: chondroitin sulfate is dissolved in deionized water, magnetically stirred at 500 rpm for 1 hour at room temperature to ensure complete dissolution, and then adjusted to pH 4.0 for the nanoparticles. Finally, the stock solution is obtained by filtration through a 0.45 μm membrane.

[0018] The present invention also provides the application of the pH-responsive fish gelatin-chondroitin sulfate nanoparticles in the preparation of a drug carrier.

[0019] This invention provides pH-responsive fish gelatin-chondroitin sulfate nanoparticles loaded with fucoxanthin. The method involves adding fucoxanthin dissolved in an organic solvent to a solution of fish gelatin-chondroitin sulfate nanoparticles, followed by ultrasonic treatment to obtain pH-responsive fish gelatin-chondroitin sulfate nanoparticles loaded with fucoxanthin. The mass ratio of fucoxanthin, fish gelatin, and chondroitin sulfate is:

[0020] Fucoxanthin 0.5-1.5 parts, fish gelatin 10-30 parts, chondroitin sulfate 5-15 parts.

[0021] More preferably, the mass ratio of fucoxanthin, fish gelatin, and chondroitin sulfate is:

[0022] 1 part fucoxanthin, 20 parts fish gelatin, and 10 parts chondroitin sulfate.

[0023] The present invention also provides a method for preparing pH-responsive fish gelatin-chondroitin sulfate nanoparticles for loading fucoxanthin, comprising the following steps:

[0024] a. Dissolve fucoxanthin in an organic solvent;

[0025] b. Add the fucoxanthin solution prepared in step a to the fish gelatin-chondroitin sulfate nanoparticle solution and sonicate to obtain pH-responsive fish gelatin-chondroitin sulfate nanoparticles loaded with fucoxanthin.

[0026] Wherein, the organic solvent in step a is anhydrous ethanol; and the ultrasonic time in step b is 10 min.

[0027] To achieve the effect of fucoxanthin being stable in gastric juice and rapidly released in intestinal juice, this invention introduces a biocompatible anionic polysaccharide—chondroitin sulfate—into fish gelatin. pH-responsive nanoparticles are prepared using a simple composite coagulation method, enabling fucoxanthin to achieve the effect of stable gastric juice and rapid release in intestinal juice. This improves the stability and bioavailability of fucoxanthin and provides a new option for the research of fucoxanthin stabilization and delivery systems. Furthermore, this invention expands the application of chondroitin sulfate in the food industry. Attached Figure Description

[0028] Figure 1 Variations of FG-CS nanoparticles under different chondroitin sulfate concentrations: (A) Particle size, PDI, and light scattering intensity; (B) Potential.

[0029] Figure 2 Effect of different fish gelatin-chondroitin sulfate ratios on phase diagrams (A) Phase diagram; (B) Apparent diagram (where the figures refer to: ■: transparent, Nanoparticles Bridges connect and condense, settlement);

[0030] Figure 3 The zeta potential of fish gelatin and chondroitin sulfate as a function of pH;

[0031] Figure 4 The binding isotherm of chondroitin sulfate titration of fish gelatin at pH 4.0;

[0032] Figure 5 Effects of different pH values ​​on FG-CS nanoparticles: (A) Particle size, PDI, and light scattering intensity; (B) Zeta potential;

[0033] Figure 6 Effects of different NaCl concentrations on FG-CS nanoparticles: (A) Particle size, PDI, and light scattering intensity; (B) Zeta potential;

[0034] Figure 7 Effects of different temperatures on FG-CS nanoparticles: (A) Particle size, PDI and light scattering intensity; (B) Zeta potential;

[0035] Figure 8 Apparent images of nanoparticles (from left to right: FG, FG-FU, FG-CS, FG-CS-FU);

[0036] Figure 9 Bar chart showing the encapsulation efficiency and loading capacity of fucoxanthin by nanoparticles;

[0037] Figure 10 Release rate of fucoxanthin in a simulated gastrointestinal tract;

[0038] Figure 11 Infrared spectrum of nanoparticles. Detailed Implementation

[0039] The following examples are used to further explain and illustrate the present invention; however, they do not constitute a limitation or restriction on the scope of the present invention.

[0040]

[0041] Example 1: Preparation of pH-responsive fish gelatin-chondroitin sulfate nanoparticles with different mass ratios according to the present invention

[0042] a. Weigh out the raw materials fish gelatin FG and chondroitin sulfate CS in the specified weight ratios;

[0043] b. Preparation of fish gelatin stock solution: The freeze-dried fish gelatin was dissolved in deionized water and magnetically stirred at 45℃ and 500rpm for 1h. After cooling to room temperature, the pH value of the nanoparticles was adjusted to 4.0. Then, the insoluble solids were removed by filtration through a 0.45μm membrane to obtain fish gelatin stock solutions of different concentrations, namely 1, 2, 3..., 10mg / mL.

[0044] c. Preparation of chondroitin sulfate stock solution: Chondroitin sulfate was dissolved in deionized water and magnetically stirred at 500 rpm for 1 h at room temperature. The pH of the nanoparticles was adjusted to 4.0. Then, the solution was filtered through a 0.45 μm membrane to obtain chondroitin sulfate stock solutions of different concentrations, namely 1, 2, 3..., 10 mg / mL.

[0045] d. Preparation and phase diagram construction of fish gelatin-chondroitin sulfate nanoparticles:

[0046] The chondroitin sulfate stock solution prepared in step c was added to the fish gelatin stock solution prepared in step b at a volume ratio of 1:1. The mixture was stirred for 30 min at room temperature using a magnetic stirrer (500 rpm) to obtain fish gelatin-chondroitin sulfate nanoparticles with different mass ratios (fish gelatin:chondroitin sulfate = 1:0, 5:1, 4:1, 3:1, 2:1, 1:1). The optimal concentration (initial concentration) of fish gelatin is 1-2 mg / mL, and the optimal ratio of fish gelatin to chondroitin sulfate is 2:1. Figure 1 The changes in FG-CS nanoparticles under different chondroitin sulfate concentrations are shown. Figure 2 The effects of different composite ratios of fish gelatin and chondroitin sulfate on the phase behavior of the system were shown.

[0047] like Figure 1 As shown, with the increase of chondroitin sulfate concentration, the light scattering intensity of the FG-CS complex increased from 0.1 to 13.6 Kcps*10. 4This indicates the formation of nanoparticles. The increase in particle size further confirms the interaction between the two. As shown in Figure A, the PDI of the FG-CS complex significantly decreased after the addition of chondroitin sulfate, reaching 0.12 at FG:CS = 2:1, demonstrating good dispersibility. Figure 2 B observed that the addition of chondroitin sulfate led to a decrease in the zeta potential of the nanoparticles, indicating that the two interacted electrostatically.

[0048] Figure 2 Observations revealed that the control group of fish gelatin or chondroitin sulfate remained transparent within the 0-10 mg / mL range. When fish gelatin and chondroitin sulfate were mixed, the composite solution exhibited three states: 1) increased turbidity leading to opacity, indicating the formation of FG-CS nanoparticles; 2) flocculation; and 3) precipitation, causing phase separation. When both gelatin and chondroitin sulfate concentrations were 1 mg / mL, precipitation occurred due to charge neutralization. When the gelatin concentration was 2 mg / mL, increasing the chondroitin sulfate concentration resulted in a transition from transparent solution, nanoparticles, precipitation, flocculation, back to nanoparticle state. When the gelatin concentration exceeded 2 mg / mL, increasing the chondroitin sulfate concentration resulted in a transition from solution to nanoparticles, flocculation, and precipitation. The experimental results demonstrate that the optimal fish gelatin-chondroitin sulfate ratio of 2:1 is sufficient to form stable nanoparticles.

[0049] Example 2: Screening of preparation parameters and performance testing of pH-responsive fish gelatin-chondroitin sulfate nanoparticles of the present invention.

[0050] I. Parameter Screening Experiment: Determination of the Optimal pH for Nanoparticles

[0051] With both gelatin and chondroitin sulfate stock solutions at a concentration of 1 mg / mL, the pH was adjusted to 2.0-10.0. The zeta potential was measured using a Malvern particle size analyzer at 25°C, and the results are as follows: Figure 3 .

[0052] The potential difference between fish gelatin and chondroitin sulfate is greatest at pH 4.0. Therefore, in order to achieve the strongest interaction between the two, the pH of fish gelatin and chondroitin sulfate is first adjusted to 4.0, and then they are mixed to prepare nanoparticles.

[0053] II. Performance Testing of pH-Responsive Fish Gelatin-Chondroitin Sulfate Nanoparticles

[0054] 1. The interaction between fish gelatin and chondroitin sulfate – isothermal titration calorific value (ITC)

[0055] First, fish gelatin, chondroitin sulfate, and deionized water (pH 4.0) were degassed for 20 minutes. Then, the degassed deionized water was added to the reference cell. The sample cell was filled with degassed fish gelatin stock solution, and chondroitin sulfate stock solution was titrated using a syringe. The sample containing chondroitin sulfate titrated with deionized water was used as a blank. When processing the data, the data points for chondroitin sulfate titrated with deionized water needed to be subtracted from the data points for chondroitin sulfate titrated with gelatin. Finally, graphs were plotted using Origin 8.0 software. Table 1 shows the thermodynamic parameters of the binding of chondroitin sulfate and gelatin. Figure 4 The binding isotherm of chondroitin sulfate titration of gelatin at pH 4.0.

[0056] from Figure 4 An approximately S-shaped saturation curve was observed. After the 17th injection of chondroitin sulfate, the curve began to flatten out, which can be attributed to the reduced amount of free protein in the reaction chamber. As a result, the fish gelatin molecules gradually bound to the chondroitin sulfate and approached saturation, entering a thermodynamically stable state (approaching 0).

[0057] Table 1 lists the thermodynamic parameters obtained after fitting. The stoichiometry n = 1.596 indicates that 1.596 chondroitin sulfate molecules bind to one gelatin molecule at pH 4.0. The negative ΔH value indicates that the binding of gelatin and chondroitin sulfate is an exothermic reaction, primarily driven by enthalpy. The exothermic reaction and ΔS > 0 (entropy increase effect) further result in a negative ΔG value for this process, exhibiting an enthalpy-entropy compensation-driven process dominated by enthalpy, confirming the spontaneity of the reaction between gelatin and chondroitin sulfate, which is beneficial to the aggregation of the complex.

[0058] Table 1. Thermodynamic parameters of chondroitin sulfate combined with gelatin

[0059]

[0060]

[0061] 2. Study on the stability of FG-CS nanoparticles by pH

[0062] The pH of the nanoparticles was adjusted to 2.0-8.0, and then the changes in particle size and potential were measured using a Malvern particle size analyzer. The results are as follows: Figure 5 .

[0063] pH affects the degree of dissociation of the amino and carboxyl groups on the side chains of gelatin and chondroitin sulfate, thus influencing the degree of interaction between them. Figure 5 As can be seen from A, when the pH is between 6.0 and 8.0, a PDI higher than 0.3 and a light scattering intensity lower than 1 Kcps*10⁴ indicate partial dissociation of the complex. At pH 5.0, the complex undergoes visible aggregation, with the particle size rapidly increasing to 15046 nm. This can be determined by… Figure 5The low charge of the complex in B leads to relatively weak electrostatic repulsion between the complexes, and the formation of insoluble protein-polysaccharide complexes can be explained by this.

[0064] FG-CS nanoparticles exhibit stability at pH 2.0 but dissociate at pH 7.0. Therefore, it is predicted that FG-CS nanoparticles can be used to protect active substances, achieving stability in gastric juice and rapid release from intestinal juice.

[0065] 3. Study on the stability of FG-CS nanoparticles by salt ions

[0066] NaCl solution (3M) was added to nanoparticles prepared in different mass ratios as in Example 1 and mixed thoroughly. The final NaCl concentrations in the composites were 0, 50, 100, 150, 200, and 250 mM, respectively. The changes in particle size and potential were measured using a Malvern particle size analyzer, and the results are as follows: Figure 6 .

[0067] Electrostatic interaction is one of the main interaction forces promoting the binding of gelatin and chondroitin sulfate. Figure 6 In study A, it was observed that the amount of NaCl added significantly affected the particle size of FG-CS nanoparticles. With increasing NaCl concentration, the absolute value of the zeta potential gradually decreased. Specifically, when the NaCl concentration in the system was between 50-100 mM, a sudden increase in particle size and PDI led to the aggregation of FG-CS nanoparticles. This can be attributed to… Figure 6 The zeta potential in B is close to 0, which can be explained by the electrostatic shielding effect of salt ions.

[0068] In addition to electrostatic interactions, non-electrostatic interactions such as hydrogen bonds, hydrophobic interactions, or van der Waals forces may also be involved. Furthermore, since the surface of FG-CS nanoparticles is mainly composed of gelatin molecular chains, there are not only electrostatic interactions between FG-CS composite nanoparticles, but also hydrophobic interactions that serve to stabilize the complex formed by the two.

[0069] 4. Study on the effect of temperature on the stability of FG-CS nanoparticles

[0070] The temperature stability of nanoparticles was evaluated by treating them at storage temperatures (4℃ and 25℃), incubation temperatures (50℃), pasteurization temperatures (75℃), and boiling temperatures (99℃) for 30 min, respectively. After cooling to room temperature, the changes in particle size and potential of the treated nanoparticles were measured using a Malvern particle size analyzer. The results are as follows: Figure 7 .

[0071] As shown in the figure, FG-CS nanoparticles maintained good dispersibility with relatively stable particle size and light scattering intensity after being treated at storage temperatures (4 and 25℃), incubation temperatures (50℃), and pasteurization temperatures (75℃) for 30 min. Even with increasing temperature, the PDI decreased slightly, indicating that appropriate heating can make the fish gelatin and chondroitin sulfate bind more tightly. However, when heated at 100℃ for 30 min, the particle size increased to over 400 nm, and the zeta potential decreased slightly, indicating that slight thermal aggregation of the particles occurred.

[0072] Overall, FG-CS nanoparticles exhibit excellent temperature stability, maintaining good stability within 75℃ without aggregation or dissociation.

[0073] In summary, ITC analysis indicates that the binding of gelatin and chondroitin sulfate is spontaneous, exothermic, and enthalpy-driven. Electrostatic interactions, hydrogen bonds, and hydrophobic interactions can coexist in the nanoparticles formed by gelatin, chondroitin sulfate, and fucoxanthin. Stability analysis shows that FG-CS nanoparticles exhibit good temperature stability. They are stable at pH 2.0 but dissociate at pH 7.0, suggesting that the encapsulation of fucoxanthin in FG-CS nanoparticles may achieve the effect of stable gastric juice and rapid release from intestinal juice.

[0074] Example 3: Preparation of pH-responsive fish gelatin-chondroitin sulfate nanoparticles loaded with fucoxanthin according to the present invention.

[0075] To load the bioactive compound, fucoxanthin was dissolved in ethanol at a concentration of 9 mg / mL. While sonicating, 34 μL of the fucoxanthin solution was added dropwise to 6 mL of the nanoparticle solution. Sonication was continued for 10 min, and this solution was named FG-CS-FU. Following the same procedure, fucoxanthin-loaded nanoparticles were prepared using gelatin alone as a control, named FG-FU. The encapsulation efficiency (EE) and loading capacity (LC) of the nanoparticles for fucoxanthin were calculated using the following formulas:

[0076]

[0077]

[0078] The relevant properties of the fucoxanthin nanoparticles are shown in Table 2, and the appearance diagram of the fucoxanthin nanoparticles is shown in the figure. Figure 8 The encapsulation efficiency and loading capacity of nanoparticles for fucoxanthin are as follows: Figure 9 .

[0079] Combine Table 2 and Figure 8The gelatin solution alone is clear and transparent, with a light scattering intensity of only 100 Kcps, indicating that the gelatin solution alone is not composed of nanoparticles. When combined with fucoxanthin, it exhibits a larger particle size and a higher light scattering intensity (9.39 Kcps * 10⁻¹⁰). 4 This indicates that a self-assembly reaction occurred to form FG-FU nanoparticles.

[0080] like Figure 9 The results showed that FG-CS nanoparticles had a higher encapsulation efficiency for fucoxanthin than gelatin alone, increasing from 62% to 77%, with the binding sites on the chondroitin sulfate surface playing a synergistic role. However, the LC of FG-FU was slightly higher than that of FG-CS-FU nanoparticles, which is because the smaller the total amount of the complex, the higher the LC value.

[0081] Table 2 Measurement Results

[0082]

[0083]

[0084] Example 4: Preparation of pH-responsive fish gelatin-chondroitin sulfate nanoparticles loaded with fucoxanthin

[0085] Dissolve 20 mg of freeze-dried fish gelatin in 10 mL of deionized water and stir magnetically at 45 °C and 500 rpm for 1 h. After cooling to room temperature, adjust the pH to 4.0 and then filter through a 0.45 μm membrane to remove insoluble solids to obtain a 2 mg / mL fish gelatin stock solution.

[0086] Dissolve 10 mg of chondroitin sulfate in 10 mL of deionized water and stir magnetically at 500 rpm for 1 h at room temperature to ensure complete dissolution. Adjust the pH to 4.0 and then filter through a 0.45 μm membrane to obtain a 1 mg / mL chondroitin sulfate stock solution.

[0087] Chondroitin sulfate stock solution was added to fish gelatin stock solution at a 1:1 volume ratio. The mixture was stirred at room temperature with a magnetic stirrer (500 rpm) for 30 min to ensure complete reaction, yielding fish gelatin-chondroitin sulfate nanoparticles (FG-CS) at a mass ratio of 2:1. 6 mL of this nanoparticle solution was used for further investigation. Fucoxanthin was dissolved in ethanol at a concentration of 9 mg / mL. While sonicating, 34 μL of the fucoxanthin solution was added dropwise to 6 mL of the nanoparticle solution. Sonication was continued for 10 min, and this mixture was named FG-CS-FU.

[0088] Example 5: Determining the gastrointestinal stability of nanoparticles using an in vitro simulated gastrointestinal tract method.

[0089] The gastrointestinal stability of nanoparticles was determined by simulating the gastrointestinal tract in vitro. Particle size and release amount were measured after each stage of sample exposure.

[0090] Stomach phase (SGF): The pH of the sample was adjusted to 2.0 with 6M HCl, and pepsin was added to make the enzyme-to-solvent ratio 1:50. The mixture was incubated in a magnetic stirrer at 100 rpm and 37°C for 120 min to simulate gastric digestion.

[0091] Small intestinal phase (SIF): The pH of the sample obtained from the above gastric phase was adjusted to 7.0 with 1M NaOH, and trypsin was added to make the enzyme-to-solvent ratio 1:25. The intestinal digestion was simulated by incubation at 100 rpm and 37°C for 120 min in a magnetic stirrer. Figure 10 The release rate of fucoxanthin in a simulated gastrointestinal tract.

[0092] like Figure 10 During the exposure of nanoparticles to SGF, gelatin wall material alone released approximately 52% of fucoxanthin. This was primarily related to the hydrolysis of gelatin, specifically depending on the pH of the stomach and the digestion by pepsin. Nanoparticles interacting via electrostatics released only 13%. In the simulated SIF process, the neutral environment of the intestinal fluid promoted the dissociation of gelatin and chondroitin sulfate, thereby exposing fucoxanthin. This fucoxanthin then came into contact with pancreatic enzymes and was enzymatically hydrolyzed, releasing more fucoxanthin than gelatin alone. Figure 5 This can be explained by pH stability.

[0093] Example 6: Verification Test of Nanoparticles Formed from Gelatin, Chondroitin Sulfate, and Fucoxanthin of the Present Invention

[0094] KBr particles containing 0.1% nanoparticles were placed in a mortar and thoroughly ground and pressed into tablets under heat and light irradiation at 4000-400 cm⁻¹. -1 The sample was scanned using a Fourier transform infrared spectrometer (FTIR) within the specified range, and the results are as follows: Figure 11 .

[0095] like Figure 11 As shown, when fucoxanthin or chondroitin sulfate is mixed with gelatin, the absorption peak in the 3100-3500 cm⁻¹ range broadens, indicating that processes such as fucoxanthin loading and polysaccharide adsorption lead to hydrogen bond formation. Furthermore, comparing the FTIR spectra of FG-FU and FG-CS-FU, the latter shows a higher intensity, indicating that the addition of chondroitin sulfate results in stronger hydrogen bond interactions in the system. The characteristic peaks of amide I and amide II in gelatin are at 1637 cm⁻¹, respectively. -1 and 1546cm -1Comparison with FG-CS spectra revealed certain differences in the characteristic peaks of amide bonds, indicating that electrostatic interaction is another force between gelatin and chondroitin sulfate. Free fucoxanthin is almost insoluble in water, while fucoxanthin can be dispersed in solutions of gelatin and FG-CS nanoparticles, suggesting hydrophobic interactions between gelatin and fucoxanthin. These results indicate that electrostatic interactions, hydrogen bonds, and hydrophobic interactions can exist in nanoparticles formed from gelatin, chondroitin sulfate, and fucoxanthin.

[0096] In summary, FG-CS nanoparticles exhibit good temperature stability. FG-CS nanoparticles demonstrate a higher encapsulation efficiency for FU compared to FG alone, increasing from 62% to 77%. FG-CS-FU nanoparticles are resistant to gastric acid and can rapidly release FU in the intestine.

Claims

1. A pH-responsive fish gelatin-chondroitin sulfate nanoparticle, characterized in that: It is made from fish gelatin and chondroitin sulfate in the following mass ratio: 2 parts fish gelatin, 1 part chondroitin sulfate; Its preparation method includes the following steps: a. Weigh out the raw materials fish gelatin and chondroitin sulfate in the specified mass ratio; b. Preparation of fish gelatin stock solution: Lyophilized fish gelatin was dissolved in deionized water and magnetically stirred for 1 hour at 45°C and 500 rpm. After cooling to room temperature, the pH of the nanoparticles was adjusted to 4. Then, 0.45... μ m-membrane filtration removes insoluble solids to obtain fish gelatin stock solution with a concentration of 2 mg / mL; c. Preparation of chondroitin sulfate stock solution: Chondroitin sulfate was dissolved in deionized water and magnetically stirred at 500 rpm for 1 hour at room temperature to adjust the pH of the nanoparticles to 4. Then, 0.45... μ m-membrane filtration was used to obtain chondroitin sulfate stock solution at a concentration of 1 mg / mL; d. Preparation of fish gelatin-chondroitin sulfate nanoparticles: The chondroitin sulfate stock solution prepared in step c was added to the fish gelatin stock solution prepared in step b at a volume ratio of 1:

1. The mixture was stirred at 500 rpm for 30 min at room temperature using a magnetic stirrer to obtain fish gelatin-chondroitin sulfate nanoparticles.

2. A method for preparing pH-responsive fish gelatin-chondroitin sulfate nanoparticles as described in claim 1, characterized in that: It includes the following steps: a. Weigh out the raw materials fish gelatin and chondroitin sulfate in the specified mass ratio; b. Preparation of fish gelatin stock solution: Lyophilized fish gelatin was dissolved in deionized water and magnetically stirred for 1 hour at 45°C and 500 rpm. After cooling to room temperature, the pH of the nanoparticles was adjusted to 4. Then, 0.45... μ m-membrane filtration removes insoluble solids to obtain fish gelatin stock solution with a concentration of 2 mg / mL; c. Preparation of chondroitin sulfate stock solution: Chondroitin sulfate was dissolved in deionized water and magnetically stirred at 500 rpm for 1 hour at room temperature to adjust the pH of the nanoparticles to 4. Then, 0.45... μ m-membrane filtration was used to obtain chondroitin sulfate stock solution at a concentration of 1 mg / mL; d. Preparation of fish gelatin-chondroitin sulfate nanoparticles: The chondroitin sulfate stock solution prepared in step c was added to the fish gelatin stock solution prepared in step b at a volume ratio of 1:

1. The mixture was stirred at 500 rpm for 30 min at room temperature using a magnetic stirrer to obtain fish gelatin-chondroitin sulfate nanoparticles.

3. The application of the pH-responsive fish gelatin-chondroitin sulfate nanoparticles according to claim 1 in the preparation of a drug carrier.

4. A pH-responsive fish gelatin-chondroitin sulfate nanoparticle for loading fucoxanthin, characterized in that: It involves adding fucoxanthin dissolved in an organic solvent to the fish gelatin-chondroitin sulfate nanoparticle solution described in claim 1, followed by ultrasonic treatment to obtain pH-responsive fish gelatin-chondroitin sulfate nanoparticles loaded with fucoxanthin, wherein the mass ratio of fucoxanthin, fish gelatin, and chondroitin sulfate is: 1 part fucoxanthin, 20 parts fish gelatin, and 10 parts chondroitin sulfate.

5. A method for preparing pH-responsive fish gelatin-chondroitin sulfate nanoparticles for loading fucoxanthin as described in claim 4, characterized in that: It includes the following steps: a. Dissolve fucoxanthin in an organic solvent; b. Add the fucoxanthin solution prepared in step a to the fish gelatin-chondroitin sulfate nanoparticle solution and sonicate for 8-12 minutes to obtain pH-responsive fish gelatin-chondroitin sulfate nanoparticles loaded with fucoxanthin.

6. The method for preparing pH-responsive fish gelatin-chondroitin sulfate nanoparticles loaded with fucoxanthin according to claim 5, characterized in that: The organic solvent mentioned in step a is anhydrous ethanol; the ultrasonic time mentioned in step b is 10 min.

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