Hydroxyapatite-artificial bear gall powder nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of hydroxyapatite-artificial bear gall powder nanoparticles, the preparation method comprises the steps of preparing hydroxyapatite nanoparticles and loading artificial bear gall powder, the preparation method is simple and easy to implement, mild in condition and suitable for drug loading, and the preparation method is suitable for large-scale industrial production. The prepared nano-particles have good biocompatibility, degradability, high safety, high stability and high aqueous medium dispersibility, and due to the stability, the nano-particles can realize long-time circulation of drugs in vivo and avoid premature removal of the drugs, so that the bioavailability of the drugs is improved; the obvious pH-responsive slow release of the artificial bear gall powder can be realized, so that the artificial bear gall powder can be used in targeted therapy; compared with other carrier materials, the adopted raw materials are low in price, do not contain metal, have great advantages in cost and environmental protection, are easier to realize large-scale industrial production, and have good application prospects.

Description

Technical Field

[0001] This invention relates to the field of artificial bear bile powder technology, specifically to a hydroxyapatite-artificial bear bile powder nanoparticle, its preparation method, and its application. Background Technology

[0002] Artificial bear bile powder is a compound drug developed based on the chemical components of natural bear bile. Its main active ingredients include various bile acids, such as ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), tauroursodeoxycholic acid (TUDCA), and taurochenodeoxycholic acid (TCDCA). It is widely used to treat various intrahepatic and extrahepatic cholestatic liver diseases, gallbladder diseases, and to prevent gallstones, serving as an effective alternative to natural bear bile. However, many active ingredients in artificial bear bile powder, especially artificial bear bile powder and CDCA, belong to BCS Class II drugs. They have poor water solubility, oral absorption varies among individuals, and higher doses are required to achieve therapeutic effects. They may also cause side effects such as gastrointestinal discomfort.

[0003] Nanoparticle drug delivery systems have attracted much attention due to their ability to improve drug solubility, enhance bioavailability, prolong in vivo circulation time, reduce toxic side effects, and achieve targeted delivery. Selecting a suitable nanocarrier is crucial for constructing efficient and safe drug nanoformulations. Hydroxyapatite (HAp), as an important calcium phosphate (CaP) material, is considered an ideal biomedical material and drug carrier due to its excellent biosafety, biodegradability, and similarity to the composition of human bones and teeth. Furthermore, hydroxyapatite nanoparticles exhibit significant pH responsiveness in acidic microenvironments (such as in inflammatory areas, tumor tissue, or intracellular lysosomes), which can accelerate carrier dissolution and promote drug release, thereby achieving targeted and on-demand delivery.

[0004] Currently, research on nano-formulations of artificial bear bile powder is relatively limited, and constructing highly efficient artificial bear bile powder nano-formulations that can be released under specific conditions using biocompatible hydroxyapatite nanocarriers remains a challenge. Therefore, developing an artificial bear bile powder nano-formulation based on hydroxyapatite nanoparticles and its simple and effective preparation method has significant clinical application prospects. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention provides hydroxyapatite-artificial bear bile powder nanoparticles, and also discloses their preparation method and applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing hydroxyapatite-artificial bear bile powder nanoparticles includes the following steps: Step S1: Preparation of hydroxyapatite nanoparticles Calcium salt and phosphate were dissolved in deionized water and hydroxyapatite was synthesized by coprecipitation. The molar ratio of calcium salt and phosphate (calculated as Ca:P) was 1.5:1 to 2.0:1. During the reaction, the pH value of the mixture was adjusted and the reaction was carried out at a certain temperature for a certain time to generate a precipitate. The precipitate was then dried to obtain hydroxyapatite nanoparticles. Step S2, Loading with artificial bear bile powder The hydroxyapatite nanoparticles prepared in step S1 were dispersed in anhydrous ethanol to obtain a nanoparticle dispersion. Then, the artificial bear bile powder solution dissolved in anhydrous ethanol was added to the nanoparticle dispersion and mixed. The mixture was shaken at a certain temperature for a certain time. The ratio of the mass of hydroxyapatite nanoparticles to the mass of artificial bear bile powder was 4:1 to 5:1. The mixture was then dried to obtain hydroxyapatite-artificial bear bile powder nanoparticles.

[0007] Furthermore, in step S1, the calcium salt is calcium chloride dihydrate, and the phosphate is diammonium hydrogen phosphate.

[0008] Furthermore, in step S1, ammonia water is added to the mixture to adjust the pH value to 9-10, and the mixture is reacted at 50-90℃ for 8-12 hours.

[0009] Furthermore, in step S1, the precipitate is vacuum dried at a temperature of 80-100℃.

[0010] Furthermore, in step S2, the oscillation temperature is 25~35℃, the oscillation speed is 150~200 rpm, the oscillation time is 24-72h, and vacuum drying or freeze drying is used. When vacuum drying is used, the vacuum temperature is 35℃ and the vacuum drying time is 2h.

[0011] A hydroxyapatite-artificial bear bile powder nanoparticle prepared by the above preparation method.

[0012] Furthermore, the physical structure of the hydroxyapatite-artificial bear bile powder nanoparticles is such that artificial bear bile powder is loaded onto the surface and pores of the porous framework formed by the aggregation of hydroxyapatite nanoparticles through physical adsorption.

[0013] Furthermore, the drug loading capacity of the nanoparticles is 15-20%.

[0014] Furthermore, the cumulative release rate of hydroxyapatite-artificial bear bile powder nanoparticles was 25-30% over 24 hours at pH 7.4 and 60-70% over 24 hours at pH 5.5.

[0015] Application of hydroxyapatite-artificial bear bile powder nanoparticles in the preparation of safe and long-acting therapeutic drugs for hepatobiliary diseases.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The hydroxyapatite nanocarrier disclosed in this invention has excellent biocompatibility and biodegradability, contains no metal components, and is highly safe as a drug carrier.

[0017] (2) The hydroxyapatite-artificial bear bile powder nanoparticles of the present invention have excellent colloidal stability, which enables the drug to circulate in the body for a long time and avoid premature clearance, thereby improving the bioavailability of the drug.

[0018] (3) The nanoparticles prepared by the present invention can efficiently load artificial bear bile powder and significantly improve its dispersibility in aqueous media. (4) The nanoparticles disclosed in this invention can achieve significant pH-responsive sustained release of artificial bear bile powder. The release is slow at physiological pH and accelerated in acidic microenvironment, which is expected to significantly improve the targeted therapeutic effect of the drug and reduce side effects.

[0019] (5) The preparation method disclosed in this invention is simple and easy to implement, with mild conditions, and is suitable for drug loading.

[0020] (6) The raw materials used in this invention are cheaper than other carrier materials and do not contain metals, which has great advantages in terms of cost and environmental protection. They are also easier to achieve large-scale industrial production and have good application prospects.

[0021] (7) The hydroxyapatite-artificial bear bile powder nanoparticles disclosed in this invention can be used in the preparation of safe and long-acting therapeutic drugs for hepatobiliary diseases, and have good practical application prospects. Attached Figure Description

[0022] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts. Wherein: Figure 1 These are the XRD patterns of the nanoparticles prepared in Examples 1-3; Figure 2 Here is a SEM image of the nanoparticles prepared in Example 1; Figure 3 Here is a SEM image of the nanoparticles prepared in Example 2; Figure 4 Here is a SEM image of the nanoparticles prepared in Example 3; Figure 5 These are optical photographs of the hydroxyapatite-artificial bear bile powder nanoparticles prepared in Examples 1-3. Detailed Implementation

[0023] It is readily understood that, based on the technical solution of this invention, those skilled in the art can propose various interchangeable structural methods and implementations without altering the essential spirit of the invention. Therefore, the following detailed embodiments and accompanying drawings are merely illustrative examples of the technical solution of this invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solution of this invention.

[0024] The technical solution of this application will now be described in detail with reference to the accompanying drawings. The description of exemplary embodiments is merely for illustrative purposes and is by no means a limitation on the invention or its application or use.

[0025] Example 1 (1) Preparation of hydroxyapatite nanoparticles: 2.21 g of CaCl₂·2H₂O was dissolved in 50 mL of deionized water to prepare solution A. 1.32 g of (NH₄)₂HPO₄ was dissolved in 50 mL of deionized water to prepare solution B. Solution B was added dropwise to solution A under magnetic stirring. Ammonia was added to adjust the pH of the mixture to 9.0, while maintaining a constant stirring speed. The reaction was carried out at 50°C for 8 hours, resulting in a white precipitate. The precipitate was collected using a centrifuge (2500 rpm, 10 minutes), washed three times with anhydrous ethanol, and vacuum dried at 80°C to obtain hydroxyapatite nanoparticles. Figure 1 The XRD pattern of hydroxyapatite-artificial bear bile powder nanoparticles is shown. Phase retrieval results indicate successful material preparation. Figure 2 As shown, hydroxyapatite nanoparticles exhibit a porous, irregular morphology composed of numerous fine particles aggregated together, forming a relatively large secondary structure. This highly porous structure and irregular surface area facilitate the physical adsorption of artificial bear bile powder into the pores and surface, thereby increasing the loading capacity.

[0026] (2) Loaded with artificial bear bile powder: Weigh 200 mg of the prepared hydroxyapatite nanoparticles and disperse them in 20 mL of anhydrous ethanol. Sonicate the mixture for 5 minutes to ensure thorough dispersion. Add 40 mg of artificial bear bile powder (pre-dissolved in 30 mL of anhydrous ethanol) to the dispersion and shake in a constant temperature shaker at 25°C for 24 hours (150 rpm) under light-protected conditions. Remove free artificial bear bile powder using a centrifuge (2500 rpm, 10 minutes).

[0027] (3) Drying: The precipitate was placed in a vacuum drying oven and dried at 35°C for 2 hours to obtain hydroxyapatite-artificial bear bile powder nanoparticles.

[0028] The drug loading of artificial bear bile powder in the nanoparticles obtained under these preparation conditions was determined to be approximately 15.2% ± 0.1 (w / w).

[0029] (4) Effect test: ①In vitro sustained-release experiment: Weigh 100 mg of the above nanoparticles and disperse them in 2.5 mL of pH 7.4 PBS buffer and pH 5.5 PBS buffer, respectively. Place the two suspensions into pre-treated dialysis bags and seal them. Place the dialysis bags into containers containing 40 mL of the corresponding PBS buffer and incubate at 37°C in a shaker (100 rpm) for release experiments. Samples were taken at 0.5, 1, 2, 4, 8, 12, and 24 hours to analyze the amount of drug released.

[0030] The in vitro sustained-release results are as follows (cumulative release rate): In pH 7.4 PBS buffer: 0.5 hours: 2.1 ± 0.4% 1 hour: 4.3 ± 0.6% 2 hours: 7.8 ± 0.9% 4 hours: 12.5 ± 1.1% 8 hours: 18.0 ± 1.5% 12 hours: 22.4 ± 1.8% 24 hours: 25.5 ± 2.1% In pH 5.5 PBS buffer: 0.5 hours: 5.2 ± 0.6% 1 hour: 10.1 ± 1.0% 2 hours: 18.5 ± 1.4% 4 hours: 30.3 ± 2.0% 8 hours: 45.6 ± 2.6% 12 hours: 55.1 ± 3.1% 24 hours: 61.5±3.5%.

[0031] ②Stability test: Zeta potential measurement: A small amount of the nanoparticles prepared in this example were dispersed in PBS buffer at pH 7.4, and their surface charge was measured using a Zeta potential analyzer. The Zeta potential of the nanoparticles prepared in this example in PBS at pH 7.4 was measured to be -15 ± 3 mV.

[0032] Particle size stability test under serum incubation: The nanoparticles prepared in this example were dispersed in a pH 7.4 PBS buffer containing 10% fetal bovine serum (FBS) to form a suspension with a concentration of 1 mg / mL. The suspension was placed in a 37°C constant-temperature shaker. Samples were taken at 0, 1, 4, 8, 12, and 24 hours, and the changes in hydrated particle size were measured by dynamic light scattering (DLS). The results showed that the initial (0 hour) hydrated particle size was approximately 150 ± 30 nm. After incubation in the serum-containing buffer for 24 hours, the hydrated particle size was 165 ± 35 nm, with no significant increase in particle size, and no obvious precipitation was observed during incubation.

[0033] The above results demonstrate that the hydroxyapatite-artificial bear bile powder nanoparticles of this invention carry a negative surface charge at physiological pH. This electrostatic repulsion effectively resists the non-specific adsorption of serum proteins, thereby exhibiting excellent colloidal stability in a simulated blood environment. This stability is the key physical basis for achieving long-term in vivo circulation, avoiding premature clearance, and thus improving drug bioavailability.

[0034] Example 2 (1) Preparation of hydroxyapatite nanoparticles: 2.65 g of CaCl₂·2H₂O was dissolved in 50 mL of deionized water to prepare solution A. 1.32 g of (NH₄)₂HPO₄ was dissolved in 50 mL of deionized water to prepare solution B. Solution B was added dropwise to solution A under magnetic stirring. Ammonia was added to adjust the pH of the mixture to 9.5, while maintaining a constant stirring speed. The reaction was carried out at 70°C for 10 hours, resulting in a white precipitate. The precipitate was collected using a centrifuge (2500 rpm, 10 minutes), washed three times with anhydrous ethanol, and vacuum dried at 90°C to obtain hydroxyapatite nanoparticles. Figure 1 The XRD pattern of hydroxyapatite-artificial bear bile powder nanoparticles is shown. Phase retrieval results indicate successful material preparation. Figure 3 As shown, hydroxyapatite nanoparticles are mainly composed of a large number of nano-sized particles loosely aggregated into micron-sized clusters. This macroscopic dispersion and microscopic porous structure together provide abundant adsorption sites and a large drug loading space for the artificial bear bile powder.

[0035] (2) Loaded with artificial bear bile powder: Weigh 200 mg of the prepared hydroxyapatite nanoparticles and disperse them in 20 mL of anhydrous ethanol. Sonicate the mixture for 5 minutes to ensure complete dispersion. Add 45 mg of artificial bear bile powder (pre-dissolved in 30 mL of anhydrous ethanol) to the dispersion and shake in a constant temperature shaker at 30°C for 36 hours (180 rpm) under light-protected conditions. Remove free artificial bear bile powder using a centrifuge (2500 rpm, 10 minutes).

[0036] (3) Drying: The precipitate was placed in a freeze dryer and freeze-dried to obtain artificial bear bile powder / hydroxyapatite nanoparticles.

[0037] The drug loading of artificial bear bile powder in the nanoparticles obtained under these preparation conditions was determined to be approximately 17.4% ± 0.2 (w / w).

[0038] (4) In vitro sustained-release experiment: The sustained-release experimental method is the same as the in vitro sustained-release experimental procedure in Example 1.

[0039] The in vitro sustained-release results are as follows (cumulative release rate): In pH 7.4 PBS buffer: 0.5 hours: 2.5 ± 0.5% 1 hour: 5.0 ± 0.7% 2 hours: 9.2 ± 1.0% 4 hours: 14.6 ± 1.3% 8 hours: 20.8 ± 1.7% 12 hours: 24.9 ± 2.0% 24 hours: 27.2 ± 2.3% In pH 5.5 PBS buffer: 0.5 hours: 6.0 ± 0.7% 1 hour: 12.2 ± 1.2% 2 hours: 21.4 ± 1.7% 4 hours: 35.1 ± 2.3% 8 hours: 49.8 ± 2.8% 12 hours: 58.6 ± 3.3% 24 hours: 64.3±3.8%.

[0040] Example 3 (1) Preparation of hydroxyapatite nanoparticles: 2.94 g of CaCl₂·2H₂O was dissolved in 50 mL of deionized water to prepare solution A. 1.32 g of (NH₄)₂HPO₄ was dissolved in 50 mL of deionized water to prepare solution B. Solution B was added dropwise to solution A under magnetic stirring. Ammonia was added to adjust the pH of the mixture to 10.0, while maintaining a constant stirring speed. The reaction was carried out at 90°C for 12 hours, resulting in a white precipitate. The precipitate was collected using a centrifuge (2500 rpm, 10 minutes), washed three times with anhydrous ethanol, and vacuum dried at 100°C to obtain hydroxyapatite nanoparticles. Figure 1 The XRD pattern of hydroxyapatite-artificial bear bile powder nanoparticles is shown. Phase retrieval results indicate successful material preparation. Figure 4 As shown, the fine particle characteristics of hydroxyapatite nanoparticles, with their accumulation and entanglement, form a rough surface rich in pores. This nanoscale roughness and high specific surface area are key factors for the effective adsorption and high drug loading of artificial bear bile powder.

[0041] (2) Loaded with artificial bear bile powder: Weigh 200 mg of the prepared hydroxyapatite nanoparticles and disperse them in 20 mL of anhydrous ethanol. Sonicate the mixture for 5 minutes to ensure complete dispersion. Add 50 mg of artificial bear bile powder (pre-dissolved in 30 mL of anhydrous ethanol) to the dispersion and shake in a constant temperature shaker at 35°C for 72 hours (200 rpm) under light-protected conditions. Remove free artificial bear bile powder using a centrifuge (2500 rpm, 10 minutes).

[0042] (3) Drying: The precipitate was placed in a vacuum drying oven and dried at 35°C for 2 hours to obtain artificial bear bile powder / hydroxyapatite nanoparticles.

[0043] The drug loading of artificial bear bile powder in the nanoparticles obtained under these preparation conditions was determined to be approximately 19.8% ± 0.2 (w / w).

[0044] (4) In vitro sustained-release experiment: The sustained-release experimental method is the same as the in vitro sustained-release experimental procedure in Example 1.

[0045] In pH 7.4 PBS buffer: 0.5 hours: 3.0 ± 0.5% 1 hour: 6.1 ± 0.8% 2 hours: 11.5 ± 1.2% 4 hours: 17.8 ± 1.6% 8 hours: 24.3 ± 2.1% 12 hours: 27.5 ± 2.4% 24 hours: 29.8 ± 2.8% In pH 5.5 PBS buffer: 0.5 hours: 3.0 ± 0.5% 1 hour: 6.1 ± 0.8% 2 hours: 11.5 ± 1.2% 4 hours: 17.8 ± 1.6% 8 hours: 24.3 ± 2.1% 12 hours: 27.5 ± 2.4% 24 hours: 29.8 ± 2.8%.

[0046] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. A method for preparing hydroxyapatite-artificial bear bile powder nanoparticles, characterized in that, Includes the following steps: Step S1: Preparation of hydroxyapatite nanoparticles Calcium salt and phosphate were dissolved in deionized water and hydroxyapatite was synthesized by co-precipitation. The molar ratio of calcium salt to phosphate was 1.5:1 to 2.0:

1. During the reaction, the pH of the mixture was adjusted and the reaction was carried out at a certain temperature for a certain time to generate a precipitate. The precipitate was then dried to obtain hydroxyapatite nanoparticles. Step S2, Loading with artificial bear bile powder The hydroxyapatite nanoparticles prepared in step S1 are dispersed in anhydrous ethanol to obtain a nanoparticle dispersion. Then, the artificial bear bile powder solution dissolved in anhydrous ethanol is added to the nanoparticle dispersion and mixed. The mixture is then shaken at a certain temperature for a certain time. The ratio of the mass of hydroxyapatite nanoparticles to the mass of artificial bear bile powder is 4:1 to 5:

1. The mixture is then dried to obtain hydroxyapatite-artificial bear bile powder nanoparticles.

2. The method for preparing hydroxyapatite-artificial bear bile powder nanoparticles according to claim 1, characterized in that, In step S1, the calcium salt is calcium chloride dihydrate, and the phosphate is diammonium hydrogen phosphate.

3. The method for preparing hydroxyapatite-artificial bear bile powder nanoparticles according to claim 1, characterized in that, In step S1, ammonia water is added to the mixture to adjust the pH value to 9-10, and the mixture is reacted at 50-90℃ for 8-12 hours.

4. The method for preparing hydroxyapatite-artificial bear bile powder nanoparticles according to claim 1, characterized in that, In step S1, the precipitate is dried under vacuum at a temperature of 80-100℃.

5. The method for preparing hydroxyapatite-artificial bear bile powder nanoparticles according to claim 1, characterized in that, In step S2, the oscillation temperature is 25~35℃, the oscillation speed is 150~200 rpm, the oscillation time is 24-72h, and vacuum drying or freeze drying is used. When vacuum drying is used, the vacuum temperature is 35℃ and the vacuum drying time is 2h.

6. A hydroxyapatite-artificial bear bile powder nanoparticle prepared by the method described in any one of claims 1 to 5.

7. The hydroxyapatite-artificial bear bile powder nanoparticles according to claim 6, characterized in that, The physical structure of the hydroxyapatite-artificial bear bile powder nanoparticles is such that the artificial bear bile powder is loaded onto the surface and pores of the porous framework formed by the aggregation of hydroxyapatite nanoparticles through physical adsorption.

8. The hydroxyapatite-artificial bear bile powder nanoparticles according to claim 6, characterized in that, The drug loading of the nanoparticles is 15-20%.

9. The hydroxyapatite-artificial bear bile powder nanoparticles according to claim 6, characterized in that, The nanoparticles exhibit a cumulative release rate of 25-30% over 24 hours at pH 7.4 and 60-70% over 24 hours at pH 5.

5.

10. The use of the hydroxyapatite-artificial bear bile powder nanoparticles according to claim 6 in the preparation of safe and long-acting therapeutic drugs for hepatobiliary diseases.

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