A method for synthesizing calcium phosphate nanoparticle-encapsulated polypeptide drug molecules using solvent phase separation

Abstract

The present application relates to a kind of synthesis calcium phosphate nanoparticles encapsulated polypeptide drug molecules using solvent phase separation method, belong to drug molecule encapsulation and nanomedicine preparation field.In the reaction system of the present application, sodium phosphate is simultaneously as solvent phase separation agent, and the raw material of synthesis, simplify the reaction system of polypeptide molecule and calcium phosphate nanoparticles synthesis and encapsulation.This method is simple, and the substance involved in reaction is less and all has biocompatibility, reaction time is shorter, can efficiently synthesize calcium phosphate nanoparticles encapsulated polypeptide drug molecules, by controlling synthesis parameter, the synthesis ratio determined by the present application can ensure the morphology of particle and certain encapsulation rate.The present application can be used as a kind of safer, more efficient platform technology, and various polypeptides are encapsulated in calcium phosphate nanoparticles, and calcium phosphate nanoparticles can also protect polypeptide molecules from the damage of harsh environment, and the present application is expected to be synthesized in large scale in the future and be used for in vivo drug delivery.

Description

Technical Field

[0001] This invention relates to a method for synthesizing calcium phosphate nanoparticles to encapsulate polypeptide drug molecules using solvent phase separation, belonging to the field of drug molecule encapsulation and nanomedicine preparation. Background Technology

[0002] Peptide drugs are short-chain molecules composed of amino acids, typically consisting of 20 to 50 amino acid residues. Their molecular weight falls between that of small-molecule chemical drugs and protein drugs, effectively bridging the gap between the two and thus becoming a new class of drugs. Peptide drugs possess high specificity, low toxicity, and good biocompatibility, and can target specific cellular or molecular pathways, showing great potential in the treatment of various diseases. Particularly in the treatment of diabetes, many novel peptide drugs have emerged, such as liraglutide, semaglutide, and tirzepatide, demonstrating certain effects in the clinical control and treatment of diabetes.

[0003] However, peptide drugs also face some challenges, among which the problems of easy enzymatic degradation and poor stability in vivo are particularly prominent. To address this issue, researchers have adopted various methods to improve the stability of peptide drugs. One such method is the nanoscale encapsulation of peptide drug molecules to enhance molecular stability and prolong the duration of drug action.

[0004] Among numerous nanomaterials used for encapsulating peptide drug molecules, calcium phosphate (CaP) offers significant advantages due to its high biocompatibility and biodegradability. The controllable solubility and biocompatibility of calcium phosphate allow for gradual degradation in the biological environment, ensuring the safe metabolism of non-toxic degradation products and supporting the sustained release of therapeutic agents in drug delivery applications. Currently, the most common method for synthesizing calcium phosphate-encapsulated peptide drugs is the mineralization method, which utilizes the deposition of calcium ions on peptide molecules, ultimately precipitating them as nanoparticles. However, this method has a long reaction time, and more importantly, it often requires multiple auxiliary substances in the reaction system, making the reaction system complex and unfavorable for subsequent purification and in vivo application. Summary of the Invention

[0005] The technical problem solved by this invention is to propose a method for synthesizing calcium phosphate nanoparticles to encapsulate peptide drug molecules using solvent phase separation. This method enables the rapid encapsulation of peptide drug molecules into calcium phosphate nanoparticles in a simple reaction system for subsequent drug delivery and treatment. Traditional methods for encapsulating peptide drug molecules with calcium phosphate involve complex reaction systems, often requiring multiple auxiliary substances that are difficult to completely remove during subsequent purification. In the reaction system of this invention, sodium phosphate, being readily soluble in water but insoluble in ethanol, serves both as a phase-separating agent and a raw material for synthesizing calcium phosphate nanoparticles, maximizing the simplification of the reaction system and facilitating subsequent product purification. Furthermore, all substances used in the synthesis method of this invention possess strong biocompatibility, fundamentally ensuring the safety of the drug in vivo. Calcium phosphate nanoparticles effectively protect peptides and improve their stability. Similarly, the calcium phosphate nanoparticles synthesized in this invention effectively protect peptide drugs from environmental damage, such as damage from alkaline environments or the presence of oxidizing agents. The reaction time for encapsulating peptide drug molecules in calcium phosphate nanoparticles is typically long, resulting in low synthesis efficiency and hindering large-scale production. The synthesis method proposed in this invention can encapsulate peptide drug molecules in calcium phosphate nanoparticles more quickly. In traditional methods for synthesizing calcium phosphate-encapsulated peptide molecules, such as mineralization methods, the reaction time is generally 3 hours or even more than 12 hours, while the reaction system of this invention can complete the reaction in only 1 hour, significantly improving the synthesis efficiency. By adjusting the synthesis parameters, the synthesis ratio determined in this invention can ensure a certain encapsulation rate while maintaining the particle morphology. Furthermore, this invention can serve as a safer and more efficient platform technology for encapsulating peptide molecules in calcium phosphate nanoparticles, and holds promise for large-scale synthesis and in vivo drug delivery in the future.

[0006] The specific technical solution proposed in this invention is: a method for synthesizing calcium phosphate nanoparticles to encapsulate polypeptide drug molecules using solvent phase separation, the specific steps of which are as follows:

[0007] Step (1): Add a mixed aqueous solution of sodium phosphate (Na3PO4) (50-100 mM) and peptide to an ethanol solution containing thickener, and vortex rapidly and let stand to induce solvent phase separation of water and ethanol to form nanodroplets.

[0008] Step (2): Add calcium chloride (CaCl2) (0.5-1.5 M) ethanol solution to the solvent-phase separated system to generate calcium phosphate nanoparticles (peptide@CaP) encapsulating peptide drug molecules (such as smegglutide, liraglutide, tesipatide and other diabetes treatment peptide drugs).

[0009] Step (3): After centrifuging the solution after the reaction, the supernatant was removed to obtain the product, which was then washed and purified with pure water multiple times to finally obtain purified peptide@CaP.

[0010] Preferably, the method for inducing solvent phase separation to form nanodroplets in step (1) is to prepare 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%) and mix 200 μL of sodium phosphate aqueous solution (70 mM) and 30 μL of peptide aqueous solution (10 mg / mL) in advance. The mixed sodium phosphate-peptide aqueous solution is quickly added to the hydroxypropyl cellulose ethanol solution and the system is vortexed for 5 s. After vortexing, the reaction system is allowed to stand for 1 min to complete the water-ethanol solvent phase separation and form nanodroplets.

[0011] Preferably, the synthesis of peptide@CaP in step (2) is specifically carried out by preparing a 1 M calcium chloride ethanol solution in advance, and after the standing in step (1), quickly adding 100 μL of calcium chloride ethanol solution (1 M) to the solvent phase separation system under vortex. After the addition is completed, the reaction system is placed in a 30 ℃ metal bath and allowed to stand for 1 h.

[0012] Preferably, the product purification process described in step (3) involves centrifuging the mixed solution after reaction at 14000 rpm for 15 min, discarding the supernatant, dispersing the precipitated product in pure water and continuing to centrifuge, repeating this process three times to achieve the purpose of washing and purifying the product. The purified product can be dispersed in a specific solvent or dried for subsequent characterization or application.

[0013] Preferably, the synthesis steps are as follows:

[0014] Step (1): Take 1 mL of hydroxypropyl cellulose ethanol solution (1 wt%), mix 200 μL of sodium phosphate aqueous solution (70 mM) and 30 μL of peptide aqueous solution (10 mg / mL) and add it quickly, and vortex for 5 s. After vortexing, let the reaction system stand for 1 min to achieve water-ethanol solvent phase separation and form nanodroplets.

[0015] Step (2): Add 100 μL of calcium chloride ethanol solution (1M) to the reaction system after it has been allowed to stand under a vortex, and let it stand in a metal bath at 30 °C for 1 h to synthesize peptide@CaP;

[0016] Step (3): After the mixed solution was allowed to stand for reaction, it was centrifuged at 14000 rpm for 15 min and the supernatant was removed to obtain the product precipitate. The product was then dispersed in pure water and centrifuged and washed three times to obtain the purified peptide@CaP.

[0017] A method for synthesizing calcium phosphate nanoparticles encapsulating peptide drug molecules using solvent phase separation includes:

[0018] Step (1): Inducing water-ethanol solvent phase separation and forming nanodroplets;

[0019] Step (2): Synthesize peptide@CaP based on the formation of nanodroplets by solvent phase separation;

[0020] Step (3): Purify the product peptide@CaP with pure water.

[0021] The beneficial effects of this invention are:

[0022] (1) A platform technology for the rapid synthesis of calcium phosphate nanoparticles and encapsulation of peptide drug molecules under a simple reaction system has been established. Under this technology, various peptide drug molecules can be encapsulated in calcium phosphate nanoparticles, which has high versatility. In the reaction system of this invention, due to the property that sodium phosphate is easily soluble in water but insoluble in ethanol, it serves as both a phase-separating agent in the water-ethanol solvent system and a raw material for the synthesis of calcium phosphate nanoparticles, which simplifies the reaction system for the synthesis of calcium phosphate nanoparticles and the encapsulation of peptide drug molecules to the greatest extent. This method has a simple reaction process, involves fewer substances in the reaction system, all of which have strong biocompatibility, and has a short reaction time, which can efficiently synthesize calcium phosphate nanoparticles (peptide@CaP) encapsulated with peptide drug molecules. By adjusting the synthesis parameters, the synthesis ratio determined by this invention can ensure a certain encapsulation rate while ensuring the particle morphology. The calculated peptide encapsulation rates are all above 30%.

[0023] (2) Calcium phosphate nanoparticles are used as a protective layer to protect peptide drug molecules, thereby improving the stability of peptide molecules. The calcium phosphate nanoparticles encapsulating peptide drug molecules obtained by the synthesis method of the present invention can effectively protect peptide molecules from damage by alkaline environments, strong oxidizing environments, etc., while the peptide drug molecules can be released within a certain period of time.

[0024] (3) Unlike other techniques for synthesizing calcium phosphate-encapsulated peptide drug molecules, which involve complex reaction systems and long reaction times, this invention simplifies the reaction system to the greatest extent possible, ensuring that calcium phosphate nanoparticles encapsulated with peptide drug molecules can be synthesized easily, while having a shorter reaction time and higher synthesis efficiency.

[0025] (4) As can be seen from Comparative Example 1, the encapsulation efficiency of liraglutide is approximately 25%, which is lower than that of Example 2. Transmission electron microscopy revealed a less uniform product morphology, with a large number of micron-sized aggregates compared to Example 2, indicating that the morphology of the product synthesized using PVP as a thickener is highly uncontrollable. Figure 8 b).

[0026] (5) As can be seen from Comparative Example 2, the product observed by transmission electron microscopy, compared with Example 2, exhibits an irregular granular morphology and contains a large amount of unformed flocculent material. Figure 9 (b) indicates that the product synthesized using a lower concentration of sodium phosphate solution has a poorer morphology.

[0027] (6) Comparative Example 3 shows that the encapsulation efficiency of liraglutide is approximately 16%, lower than that of Example 2. Transmission electron microscopy revealed that the product consists of spherical hollow nanoparticles. Figure 10 b). This demonstrates that the ability to synthesize CaP-encapsulated peptide drug molecules using a higher concentration of sodium phosphate solution is inferior to that in Example 2.

[0028] (7) Comparative Example 4 shows that the encapsulation efficiency of liraglutide is approximately 27%, lower than that of Example 2. Transmission electron microscopy revealed that the product consists of spherical hollow nanoparticles. Figure 11 b). This demonstrates that the ability to synthesize CaP-encapsulated peptide drug molecules using a lower concentration of calcium chloride ethanol solution is inferior to that in Example 2.

[0029] Example 2 showed an encapsulation efficiency of approximately 31% for liraglutide. Transmission electron microscopy and scanning electron microscopy revealed that the product consisted of relatively uniform spherical hollow nanoparticles. Figure 3 (This is the best embodiment.)

[0030] The calcium phosphate nanoparticles in Example 2 effectively protect peptide molecules from damage in an alkaline environment. Simultaneously, transmission electron microscopy was used to observe Lira.@CaP nanoparticles treated with sodium carbonate solution. Figure 13 c) It can be observed that its nanostructure was not damaged. Liraglutide encapsulated in calcium phosphate nanoparticles also exhibited a strong peptide UV absorption signal under strong oxidative conditions, indicating that calcium phosphate nanoparticles can effectively protect peptide molecules from damage by strong oxidative environments. Furthermore, transmission electron microscopy observation of Lira.@CaP (after treatment with hydrogen peroxide solution) Figure 14 c) It can be seen that its nanostructure has not been damaged.

[0031] Lira.@CaP (the product of Example 2) was dispersed in a Glycine-HCl buffer solution (pH = 2.2) for a release experiment. During the release process, the product precipitate gradually dissolved and completely dissolved within approximately 48 hours. Figure 15 a) The UV absorption signal of the solution after product digestion was detected using a UV-Vis spectrophotometer, and a significant peptide molecule absorption peak was found. Figure 15(b) demonstrates that peptide drug molecules encapsulated by calcium phosphate nanoparticles can be released within a certain time. Attached Figure Description

[0032] The present invention will be further described below with reference to the accompanying drawings.

[0033] Figure 1 This is a schematic diagram illustrating the mechanism of synthesizing nanoscale calcium phosphate-encapsulated peptide drug molecules using solvent phase separation.

[0034] Figure 2 This is a comparison of the UV-Vis absorption spectra of calcium phosphate nanoparticles (Lira.@CaP) encapsulating liraglutide, an aqueous solution of liraglutide, and calcium phosphate nanoparticles.

[0035] Figure 3 (a) is a transmission electron microscope image of Lira.@CaP; (b) is a scanning electron microscope image of Lira.@CaP.

[0036] Figure 4 This is a comparison of the UV-Vis absorption spectra of calcium phosphate nanoparticles (Sema.@CaP) encapsulated with semaglutide, an aqueous solution of semaglutide, and calcium phosphate nanoparticles.

[0037] Figure 5 (a) is a transmission electron microscope image of Sema.@CaP; (b) is a scanning electron microscope image of Sema.@CaP.

[0038] Figure 6 This is a comparison of the UV-Vis absorption spectra of calcium phosphate nanoparticles (Tirz.@CaP) encapsulated with tesiparatide, an aqueous solution of tesiparatide, and calcium phosphate nanoparticles.

[0039] Figure 7 (a) is a transmission electron microscope image of Tirz.@CaP; (b) is a scanning electron microscope image of Tirz.@CaP.

[0040] Figure 8 (a) is a comparison of the UV-Vis absorption spectra of Lira.@CaP (PVP) and liraglutide aqueous solution; (b) is a transmission electron microscope image of Lira.@CaP (PVP).

[0041] Figure 9 (a) is a comparison of the UV-Vis absorption spectra of Lira.@CaP (50 mM Na2PO4) and liraglutide aqueous solution; (b) is a transmission electron microscope image of Lira.@CaP (50 mM Na2PO4).

[0042] Figure 10(a) is a comparison of the UV-Vis absorption spectra of Lira.@CaP (100 mM Na2PO4) and liraglutide aqueous solution; (b) is a transmission electron microscope image of Lira.@CaP (100 mM Na2PO4).

[0043] Figure 11 (a) is a comparison of the UV-Vis absorption spectra of Lira.@CaP (0.5 M CaCl2) and liraglutide aqueous solution; (b) is a transmission electron microscope image of Lira.@CaP (0.5 M CaCl2).

[0044] Figure 12 (a) is a comparison of the UV-Vis absorption spectra of Lira.@CaP (1.5 M CaCl2) and liraglutide aqueous solution; (b) is a transmission electron microscope image of Lira.@CaP (1.5 M CaCl2).

[0045] Figure 13 (a) is a comparison of the UV-Vis absorption spectra of Lira.@CaP after treatment with sodium carbonate solution or water; (b) is a comparison of the UV-Vis absorption spectra of free liraglutide after treatment with sodium carbonate solution or water; (c) is a transmission electron microscope image of Lira.@CaP after treatment with sodium carbonate solution.

[0046] Figure 14 (a) is a comparison of the UV-Vis absorption spectra of Lira.@CaP after treatment with hydrogen peroxide solution or water; (b) is a comparison of the UV-Vis absorption spectra of free liraglutide after treatment with hydrogen peroxide solution or water; (c) is a transmission electron microscope image of Lira.@CaP after treatment with hydrogen peroxide solution.

[0047] Figure 15 (a) is a photograph of Lira.@CaP before and after digestion; (b) is a comparison of the UV-Vis absorption spectra of the digested Lira.@CaP solution and the aqueous solution of liraglutide. Detailed Implementation

[0048] Example 1

[0049] 200 μL of sodium phosphate aqueous solution (70 mM) was rapidly added to 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%). The solution was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL of calcium chloride ethanol solution (1 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction, the reaction system was centrifuged at 14000 rpm for 15 min. After removing the supernatant, the product precipitate was washed three times with pure water to obtain the product CaP. The product was then dispersed in pure water. The UV absorption signal of the product was detected using a UV-Vis spectrophotometer, but no UV absorption signal peak was found. Figure 2 , Figure 4 , Figure 6 ).

[0050] Example 2

[0051] 200 μL of sodium phosphate aqueous solution (70 mM) and 30 μL of liraglutide aqueous solution (10 mg / mL) were mixed and then rapidly added to 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%). The mixture was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL of calcium chloride ethanol solution (1 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction, the mixture was centrifuged at 14000 rpm for 15 min, the supernatant was removed, and the product precipitate was washed three times with pure water to obtain the product Lira.@CaP. The product was then dispersed in pure water. The UV absorption signal of the product was detected using a UV-Vis spectrophotometer and compared with that of the liraglutide aqueous solution. A distinct peptide molecular absorption peak was observed. Figure 2 This verified that the peptide molecule was encapsulated in CaP nanoparticles. The UV-Vis absorption curve of CaP was used as background and subtracted from the UV-Vis absorption curve of Lira.@CaP. The encapsulation efficiency of liraglutide by this method was calculated to be approximately 31% using the relationship between the intensity of the characteristic absorption peak of the liraglutide solution and concentration. Transmission electron microscopy and scanning electron microscopy revealed that the product consisted of relatively uniform spherical hollow nanoparticles. Figure 3 ).

[0052] Example 3

[0053] A mixture of 200 μL sodium phosphate aqueous solution (70 mM) and 30 μL semaglutide aqueous solution (10 mg / mL) was rapidly added to 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%). The mixture was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL calcium chloride ethanol solution (1 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction, the mixture was centrifuged at 14000 rpm for 15 min. The supernatant was removed, and the product precipitate was washed three times with pure water to obtain the product Sema.@CaP. The product was then dispersed in pure water. The UV absorption signal of the product was detected using a UV-Vis spectrophotometer and compared with that of the semaglutide aqueous solution. A distinct peptide molecular absorption peak was observed. Figure 4 The UV-Vis absorption curve of CaP was used as background and subtracted from the UV-Vis absorption curve of Sema.@CaP. The encapsulation efficiency of semaglutide by this method was calculated to be approximately 40% using the relationship between the intensity of the characteristic absorption peak of the semaglutide solution and concentration. Transmission electron microscopy and scanning electron microscopy revealed that the product consisted of relatively uniform spherical hollow nanoparticles. Figure 5 ).

[0054] Example 4

[0055] A mixture of 200 μL sodium phosphate aqueous solution (70 mM) and 30 μL tesiparatide aqueous solution (10 mg / mL) was rapidly added to 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%). The mixture was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL calcium chloride ethanol solution (1 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction was complete, the reaction system was centrifuged at 14000 rpm for 15 min. After removing the supernatant, the product precipitate was washed three times with pure water to obtain the product Tirz.@CaP, which was then dispersed in pure water.

[0056] The UV absorption signal of the product was detected using a UV-Vis spectrophotometer and compared with that of tesipatide aqueous solution, revealing a distinct absorption peak of the polypeptide molecule. Figure 6 The UV-Vis absorption curve of CaP was used as background and subtracted from the UV-Vis absorption curve of Tirz.@CaP. The encapsulation efficiency of tesipatide by this method was calculated to be approximately 32% using the relationship between the intensity of the characteristic absorption peak of the tesipatide solution and concentration. Transmission electron microscopy and scanning electron microscopy revealed that the product consisted of relatively uniform spherical hollow nanoparticles. Figure 7 ).

[0057] Comparative Example 1

[0058] 200 μL of sodium phosphate aqueous solution (70 mM) and 30 μL of liraglutide aqueous solution (10 mg / mL) were mixed and then rapidly added to 1 mL of polyvinylpyrrolidone (PVP) thickener ethanol solution (1 wt%). The mixture was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL of calcium chloride ethanol solution (1 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction, the reaction system was centrifuged at 14000 rpm for 15 min, the supernatant was removed, and the product precipitate was washed three times with pure water to obtain the product Lira.@CaP (PVP). The product was dispersed in pure water. The UV absorption signal of the product was detected using a UV-Vis spectrophotometer and compared with that of liraglutide aqueous solution, revealing a clear peptide molecular absorption peak (…). Figure 8 a). The UV-Vis absorption curve of CaP was used as background and subtracted from the UV-Vis absorption curve of Lira.@CaP (PVP). The encapsulation efficiency of liraglutide by this method was calculated to be approximately 25% using the relationship between the intensity of the characteristic absorption peak of the liraglutide solution and concentration, which is lower than the encapsulation efficiency in Example 2. Transmission electron microscopy revealed a less uniform product morphology, with a large number of micron-sized aggregates compared to Example 2, indicating that the morphology of the product synthesized using PVP as a thickener is highly uncontrollable. Figure 8 b).

[0059] Comparative Example 2

[0060] 200 μL of sodium phosphate aqueous solution (50 mM) and 30 μL of liraglutide aqueous solution (10 mg / mL) were mixed and then rapidly added to 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%). The mixture was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL of calcium chloride ethanol solution (1 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction, the mixture was centrifuged at 14000 rpm for 15 min, the supernatant was removed, and the product precipitate was washed three times with pure water to obtain the product Lira.@CaP (50 mM Na2PO4). The product was dispersed in pure water. The UV absorption signal of the product was detected using a UV-Vis spectrophotometer and compared with that of the liraglutide aqueous solution. A distinct peptide molecular absorption peak was observed. Figure 9a). The UV-Vis absorption curve of CaP was used as background and subtracted from the UV-Vis absorption curve of Lira.@CaP (50 mM Na2PO4). The encapsulation efficiency of liraglutide by this method was calculated to be approximately 35% using the relationship between the intensity of the characteristic absorption peak of the liraglutide solution and concentration, which is slightly higher than the encapsulation efficiency in Example 2. However, transmission electron microscopy revealed that the product exhibited an irregular granular morphology compared to Example 2, with a greater presence of unformed flocculent material (…). Figure 9 (b) indicates that the product synthesized using a lower concentration of sodium phosphate solution has a poorer morphology.

[0061] Comparative Example 3

[0062] A mixture of 200 μL sodium phosphate aqueous solution (100 mM) and 30 μL liraglutide aqueous solution (10 mg / mL) was rapidly added to 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%). The mixture was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL calcium chloride ethanol solution (1 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction, the system was centrifuged at 14000 rpm for 15 min. The supernatant was removed, and the product precipitate was washed three times with pure water to obtain the product Lira.@CaP (100 mM Na2PO4). The product was dispersed in pure water. The UV absorption signal of the product was detected using a UV-Vis spectrophotometer and compared with that of the liraglutide aqueous solution. A distinct peptide molecular absorption peak was observed. Figure 10 a). The UV-Vis absorption curve of CaP was used as background and subtracted from the UV-Vis absorption curve of Lira.@CaP (100mM Na2PO4). The encapsulation efficiency of liraglutide by this method was calculated to be approximately 16% using the relationship between the intensity of the characteristic absorption peak of the liraglutide solution and concentration, which is lower than the encapsulation efficiency in Example 2. Transmission electron microscopy revealed that the product consisted of spherical hollow nanoparticles. Figure 10 b). This demonstrates that the ability to synthesize CaP-encapsulated peptide drug molecules using a higher concentration of sodium phosphate solution is inferior to that in Example 2.

[0063] Comparative Example 4

[0064] 200 μL of sodium phosphate aqueous solution (70 mM) and 30 μL of liraglutide aqueous solution (10 mg / mL) were mixed and then rapidly added to 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%). The mixture was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL of calcium chloride ethanol solution (0.5 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction, the mixture was centrifuged at 14000 rpm for 15 min. After removing the supernatant, the product precipitate was washed three times with pure water to obtain the product Lira.@CaP (0.5 M CaCl2), which was then dispersed in pure water. The UV absorption signal of the product was detected using a UV-Vis spectrophotometer and compared with that of the liraglutide aqueous solution. A distinct peptide molecular absorption peak was observed. Figure 11 a). The UV-Vis absorption curve of CaP was used as background and subtracted from the UV-Vis absorption curve of Lira.@CaP (0.5M CaCl2). The encapsulation efficiency of liraglutide by this method was calculated to be approximately 27% using the relationship between the intensity of the characteristic absorption peak of the liraglutide solution and concentration, which is lower than the encapsulation efficiency in Example 2. Transmission electron microscopy revealed that the product consisted of spherical hollow nanoparticles. Figure 11 b). This demonstrates that the ability to synthesize CaP-encapsulated peptide drug molecules using a lower concentration of calcium chloride ethanol solution is inferior to that in Example 2.

[0065] Comparative Example 5

[0066] 200 μL of sodium phosphate aqueous solution (70 mM) and 30 μL of liraglutide aqueous solution (10 mg / mL) were mixed and then rapidly added to 1 mL of hydroxypropyl cellulose (HPC) thickener ethanol solution (1 wt%). The mixture was vortexed for 5 s and then allowed to stand for 1 min. Then, 100 μL of calcium chloride ethanol solution (1.5 M) was rapidly added to the mixture under vortexing, and the reaction system was placed in a metal bath at 30 °C for 1 h. After the reaction, the mixture was centrifuged at 14000 rpm for 15 min. After removing the supernatant, the product precipitate was washed three times with pure water to obtain the product Lira.@CaP (1.5 M CaCl2), which was then dispersed in pure water. The UV absorption signal of the product was detected using a UV-Vis spectrophotometer and compared with that of the liraglutide aqueous solution. A distinct peptide molecular absorption peak was observed. Figure 12a). The UV-Vis absorption curve of CaP was used as background and subtracted from the UV-Vis absorption curve of Lira.@CaP (1.5M CaCl2). The encapsulation efficiency of liraglutide by this method was calculated to be approximately 32% using the relationship between the intensity of the characteristic absorption peak of the liraglutide solution and concentration, which is similar to the encapsulation efficiency in Example 2. However, transmission electron microscopy revealed that, compared to Example 2, the product exhibited significant aggregation, with numerous micron-sized large particles and a large amount of unformed flocculent material. Figure 12 (b) indicates that the product synthesized using a higher concentration of sodium phosphate solution has a poorer morphology and a higher degree of morphological uncontrollability.

[0067] Experiment 1

[0068] Lira.@CaP (product of Example 2) dispersed in 10 μL of water was added to 200 μL of sodium carbonate solution (2 M, pH = 12) or 200 μL of water. After waiting for 10 min, the UV absorption signal of the solution was detected using a UV-Vis spectrophotometer. Figure 13 a). Simultaneously, 10 μL of liraglutide aqueous solution was added to either 200 μL of sodium carbonate solution (2 M, pH = 12) or 200 μL of water, and the UV absorption signal of the solution was detected using a UV-Vis spectrophotometer as a control group. Figure 13 b). It can be clearly observed that free liraglutide no longer exhibits characteristic UV absorption signals in the alkaline environment of sodium carbonate solution, indicating that the peptide structure has been damaged; however, liraglutide encapsulated in calcium phosphate nanoparticles still shows a strong peptide UV absorption signal in the alkaline environment, indicating that calcium phosphate nanoparticles can effectively protect peptide molecules from damage by the alkaline environment. Simultaneously, transmission electron microscopy was used to observe Lira.@CaP (…) after treatment with sodium carbonate solution. Figure 13 c) It can be seen that its nanostructure has not been damaged.

[0069] Experiment 2

[0070] Lira.@CaP (product of Example 2) dispersed in 10 μL of water was added to 200 μL of 3% hydrogen peroxide solution or 200 μL of water. After waiting for 10 min, the UV absorption signal of the solution was detected using a UV-Vis spectrophotometer. Figure 14 a). Simultaneously, 10 μL of liraglutide aqueous solution was added to either 200 μL of 3% hydrogen peroxide solution or 200 μL of water, and the UV absorption signal of the solution was detected using a UV-Vis spectrophotometer as a control group. Figure 14b). It can be clearly observed that free liraglutide no longer exhibits characteristic UV absorption signals under the strong oxidizing environment of hydrogen peroxide solution, indicating that the peptide structure has been damaged; however, liraglutide encapsulated in calcium phosphate nanoparticles still shows a strong peptide UV absorption signal under strong oxidizing conditions, indicating that calcium phosphate nanoparticles can effectively protect peptide molecules from damage by strong oxidizing environments. Simultaneously, transmission electron microscopy was used to observe Lira.@CaP (…) after treatment with hydrogen peroxide solution. Figure 14 c) It can be seen that its nanostructure has not been damaged.

[0071] Experiment 3

[0072] Lira.@CaP (the product of Example 2) was dispersed in a Glycine-HCl buffer solution (pH = 2.2) for a release experiment. During the release process, the product precipitate gradually dissolved and completely dissolved within approximately 48 hours. Figure 15 a) The UV absorption signal of the solution after product digestion was detected using a UV-Vis spectrophotometer, and a significant peptide molecule absorption peak was found. Figure 15 (b) demonstrates that peptide drug molecules encapsulated by calcium phosphate nanoparticles can be released within a certain time period.

[0073] The present invention is not limited to the specific technical solutions described in the above embodiments. All technical solutions formed by equivalent substitutions are within the scope of protection claimed by the present invention.

Claims

1. A method for synthesizing calcium phosphate nanoparticles encapsulating polypeptide drug molecules using solvent phase separation, characterized in that: Mix 200 μL of 70 mM sodium phosphate aqueous solution and 30 μL of 10 mg / mL liraglutide aqueous solution and then quickly add the mixture to 1 mL of 1 wt% hydroxypropyl cellulose thickener ethanol solution. Vortex the mixture for 5 seconds and let it stand for 1 minute. Then, quickly add 100 μL of 1 M calcium chloride ethanol solution to the mixture after it has stood under vortexing. Place the reaction system in a metal bath and react at 30°C for 1 hour. After the reaction was completed, the reaction system was centrifuged at 14000 rpm for 15 min. After removing the supernatant, the product precipitate was washed and purified three times with pure water to obtain the product Lira.@CaP.

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