Solubilization and slow release method of progesterone protein nano sustained release preparation

Abstract

The invention relates to a solubilizing and slow-release method of a progesterone protein nano sustained-release preparation, which comprises the following steps: preparing raw materials, namely preparing a framework material, an adsorption layer material, a protein carrier and a freeze-drying protective additive, preparing nanoparticles, preparing drug-loaded nanoparticles by an emulsification-ultrasonic method, and carrying out freeze-drying treatment, namely carrying out freeze-drying treatment by adopting a stepped heating freeze-drying process, so as to obtain the progesterone protein nano sustained-release preparation. The freeze-dried powder is redissolved to form a stable nano dispersion with the average particle size smaller than or equal to 100 nm, nanoparticles can be effectively prevented from gathering in the environment with physiological salt ions or serum protein, stable hydrogen bonds can be formed with protein molecules, effective protection is provided for a protein carrier in the freeze-drying process, and the stability of the preparation is improved. The progesterone protein nano sustained-release preparation has the advantages that the progesterone protein nano sustained-release preparation can inhibit the denaturation of a protein carrier, the solubility of progesterone can be obviously improved by the compound surfactant, slow release of drugs can be realized by the aid of the amphiphilic polymer, and in-vivo and in-vitro experiments show that the progesterone protein nano sustained-release preparation prepared by the aid of the method can effectively prolong the release time of the drugs and improve the bioavailability of the drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, in particular to a solubilization and sustained-release method for a progesterone protein nano sustained-release preparation. Background Art

[0002] Progesterone is an important progestogen with a wide range of clinical applications, such as treating irregular menstruation and threatened abortion. However, progesterone's poor water solubility limits the development and application of its formulations. Formulating progesterone into a protein nanoparticle sustained-release formulation can effectively improve the drug's bioavailability, achieve slow release, prolong the duration of drug action, reduce the frequency of medication use, and improve patient compliance. Currently, there are many problems in the preparation and storage of progesterone protein nano-sustained-release preparations. Among them, nanoparticles are prone to aggregation. For example, after 7 days of storage, the particle size may increase from the initial 100nm to 500nm. Especially in the presence of physiological salt ions or serum proteins, the aggregation phenomenon is more serious, which will not only affect the sustained-release effect of the drug, but may also lead to a decrease in the stability of the drug and affect its clinical efficacy. In addition, commonly used lyoprotectants such as mannitol have limitations in protecting protein carriers and cannot completely inhibit the denaturation of protein carriers, thereby reducing the quality and effectiveness of the preparation.

[0003] Therefore, it is urgent to develop a new solubilization and sustained-release method to solve the above problems. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a solubilization and sustained-release method for a progesterone protein nano-sustained-release preparation to solve the technical problems raised in the above-mentioned background technology.

[0005] The object of the present invention is to provide a solubilization and sustained-release method for a progesterone protein nano-sustained-release preparation to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for solubilizing and sustained-release of a progesterone protein nano-sustained-release preparation, comprising the following steps: Raw material preparation: including the preparation of skeleton materials, adsorption layer materials, protein carriers, and freeze-drying protective agents; Nanoparticle preparation: drug-loaded nanoparticles are prepared by emulsification-ultrasound method; Freeze-drying treatment: using step-by-step temperature freeze-drying process; Preparation molding: The freeze-dried powder is reconstituted to form a stable nano-dispersion with an average particle size of ≤100nm.

[0007] Preferably, the raw material preparation includes the preparation of the skeleton material, the preparation of the adsorption layer, the preparation of the protein carrier, the preparation of the lyophilization protective agent and the preparation of other raw materials; Select amphiphilic polymers with a molecular weight of 5000-15000Da, such as methoxy polyethylene glycol-polycaprolactone (mPEG-PCL), as the backbone material of the nanoparticles; A composite system of the nonionic surfactant Tween 80 and the anionic surfactant sodium dodecyl sulfate (SDS) was selected, with a mass ratio of Tween 80 to SDS of 3:1. At this ratio, the surface zeta potential of the nanoparticles was measured by dynamic light scattering to be -30±2 mV, and an adsorption layer with a thickness of 5±1 nm was formed. The composite surfactant was able to form a dense adsorption layer on the surface of the nanoparticles. Bovine serum albumin (BSA) was used as the protein carrier; Prepare a lyoprotectant, using a mixture of trehalose and sorbitol in a mass ratio of 2:1 to replace traditional mannitol. Trehalose and sorbitol can form hydrogen bonds with protein molecules, effectively protecting the structure and function of the protein carrier during the lyophilization process and inhibiting its denaturation. Prepare progesterone API, deionized water and other raw materials.

[0008] Preferably, the nanoparticle preparation comprises dissolving a certain amount of amphiphilic polymer, compound surfactant and progesterone in an organic solvent of dichloromethane to form an oil phase; dissolving the protein carrier in deionized water to form an aqueous phase, wherein the volume ratio of the oil phase to the aqueous phase is 1:3; Under a stirring speed of 1200±50rpm, the oil phase was added dropwise at a rate of 0.5mL / min to form a primary emulsion; The primary emulsion was transferred to a probe sonicator and sonicated for 12±1 min at a power of 250 W and a frequency of 20 kHz. The resulting nanoemulsion had a particle size distribution (PDI) of ≤0.15.

[0009] Preferably, the freeze-drying process refers to adding a freeze-protectant to the nanoemulsion, the mass concentration of the freeze-protectant is 5-10%, the nanoemulsion containing the freeze-protectant is divided into freeze-drying bottles, the pre-freezing temperature is -40 ° C, the pre-freezing time is 3-4h, the nanoemulsion is completely frozen, and vacuum freeze-drying is performed. The freeze-drying process is divided into three stages: In the first stage, the temperature was raised from -40℃ to -20℃, and the vacuum degree was maintained at 10-20Pa for 5-6h; In the second stage, the temperature is raised from -20℃ to 0℃, and the vacuum degree is maintained at 5-10Pa for 8-10h; In the third stage, the temperature is raised from 0°C to 20°C, and the vacuum degree is maintained at 2-5 Pa for 4-5 hours; The specific surface area of ​​the lyophilized nanoparticles was determined to be 35±3m² / g by the BET method, and the protein activity retention rate after reconstitution of the native conformation ratio was determined by HPLC to be ≥95%.

[0010] Preferably, the freeze-dried powder is added to physiological saline at a ratio of 1:10 (w / v), and after 30 seconds of ultrasonic treatment at 40kHz, the following is measured: Dynamic light scattering showed an average particle size of 80 ± 5 nm; Transmission electron microscopy showed that the core-shell structure was intact; The particle size change rate is less than 5% under 4°C storage conditions within 3 months; The in vitro release curve of the reconstituted preparation conforms to the Higuchi equation, and the cumulative release rate in pH 7.4 phosphate buffer is 65±5% over 24 hours and reaches more than 90% over 72 hours.

[0011] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: 1. Effectively inhibiting nanoparticle aggregation: By selecting specific amphiphilic polymers and compounded surfactants, the present invention forms a hydration layer and a compact adsorption layer on the surface of the nanoparticles, effectively preventing the nanoparticles from aggregating in the presence of physiological salt ions or serum proteins. Experimental verification shows that after 7 days of storage, the nanoparticle size can still be maintained in the range of 100-150nm, significantly improving the stability of the preparation. 2. Preventing protein carrier denaturation: A mixture of trehalose and sorbitol is used as a freeze-drying protectant, which can form stable hydrogen bonds with protein molecules, providing effective protection for the protein carrier during the freeze-drying process and inhibiting its denaturation. Testing has shown that the activity retention rate of the protein carrier is above 90%, ensuring the effectiveness of the preparation. 3. Improve drug solubilization and sustained-release effects: The compounded surfactant can significantly improve the solubility of progesterone, and the presence of the amphiphilic polymer helps to achieve slow release of the drug. In vitro and in vivo experiments show that the progesterone protein nano-sustained-release preparation prepared by the present invention can effectively prolong the release time of the drug and improve the bioavailability of the drug. DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0013] Example 1 Raw material preparation Weigh 1g of methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) with a molecular weight of 10,000Da. This amphiphilic polymer has good biocompatibility and degradability. Its hydrophilic polyethylene glycol chain segment can form a hydration layer on the surface of nanoparticles, effectively preventing aggregation between nanoparticles.

[0014] Weigh 0.6 g of Tween 80 and 0.2 g of sodium dodecyl sulfate (SDS) and mix them evenly to form a composite surfactant. Weigh 0.5 g of bovine serum albumin (BSA), which has good biocompatibility and drug loading capacity. Weigh 1g of trehalose and 0.5g of sorbitol and mix them evenly as a freeze-drying protective agent. Weigh 0.2g of progesterone API and prepare appropriate amounts of dichloromethane and deionized water. Nanoparticle preparation mPEG-PCL, composite surfactant and progesterone were dissolved in 10 mL of dichloromethane to form an oil phase; BSA was dissolved in 30 mL of deionized water to form an aqueous phase. The oil phase was slowly added dropwise to the water phase at a stirring speed of 1200 rpm to form a primary emulsion. The primary emulsion was transferred to a probe sonicator and sonicated at a power of 250 W for 12 min to form a stable nanoemulsion. Freeze-drying Add 2.25 g of freeze-dried protective agent to the nanoemulsion and stir evenly. The nanoemulsion containing the lyoprotectant was dispensed into lyophilization bottles and pre-frozen at -40°C for 3.5 h. Vacuum freeze-drying was carried out. In the first stage, the temperature was raised from -40℃ to -20℃, the vacuum degree was maintained at 15Pa, and the time was 5.5h; in the second stage, the temperature was raised from -20℃ to 0℃, the vacuum degree was maintained at 8Pa, and the time was 9h; in the third stage, the temperature was raised from 0℃ to 20℃, the vacuum degree was maintained at 3Pa, and the time was 4.5h. By optimizing the freeze-drying process, the residual moisture can be reduced and the denaturation of the protein carrier during the freeze-drying process can be avoided. Preparation molding The freeze-dried powder was reconstituted with saline to produce a progesterone protein nanoparticle sustained-release formulation. Testing showed that the initial average particle size of the nanoparticles in this formulation was 110 nm. After seven days of storage in saline at 37°C, the average particle size decreased to 130 nm. The activity retention rate of the protein carrier was 92%. Example 2 Raw material preparation Weigh 0.8g of methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) with a molecular weight of 8000Da. This amphiphilic polymer has good biocompatibility and degradability. Its hydrophilic polyethylene glycol chain segment can form a hydration layer on the surface of nanoparticles, effectively preventing aggregation between nanoparticles. Weigh 0.45 g of Tween 80 and 0.15 g of sodium dodecyl sulfate (SDS) and mix them evenly to form a composite surfactant. Weigh 0.4 g of bovine serum albumin (BSA), which has good biocompatibility and drug loading capacity. Weigh 0.8 g of trehalose and 0.4 g of sorbitol and mix them evenly as a freeze-drying protective agent. Weigh 0.15g of progesterone API and prepare appropriate amounts of dichloromethane and deionized water. Nanoparticle preparation mPEG-PCL, composite surfactant and progesterone were dissolved in 8 mL of dichloromethane to form an oil phase; BSA was dissolved in 24 mL of deionized water to form an aqueous phase. The oil phase was slowly added dropwise to the water phase at a stirring speed of 1000 rpm to form a primary emulsion. The primary emulsion was transferred to a probe sonicator and sonicated at a power of 200 W for 10 min to form a stable nanoemulsion. Freeze-drying Add 1.8 g of freeze-dried protective agent to the nanoemulsion and stir evenly. The nanoemulsion containing the lyoprotectant was dispensed into lyophilization bottles and pre-frozen at -40°C for 3 h.

[0015] Vacuum freeze-drying was carried out. In the first stage, the temperature was raised from -40℃ to -20℃, the vacuum degree was maintained at 10Pa, and the time was 5h; in the second stage, the temperature was raised from -20℃ to 0℃, the vacuum degree was maintained at 5Pa, and the time was 8h; in the third stage, the temperature was raised from 0℃ to 20℃, the vacuum degree was maintained at 2Pa, and the time was 4h. By optimizing the freeze-drying process, the residual moisture can be reduced and the denaturation of the protein carrier during the freeze-drying process can be avoided.

[0016] Preparation molding The freeze-dried powder was reconstituted with saline to produce a progesterone protein nanoparticle sustained-release formulation. Testing showed that the initial average particle size of the nanoparticles in this formulation was 105 nm. After seven days of storage in saline at 37°C, the average particle size decreased to 125 nm. The activity retention rate of the protein carrier was 91%. Comparative Example 1 (using traditional mannitol as freeze-drying protective agent) Raw material preparation Weigh 1 g of methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) with a molecular weight of 10,000 Da. Weigh 0.6 g of Tween 80 and 0.2 g of sodium dodecyl sulfate (SDS) and mix them evenly to form a composite surfactant. Weigh 0.5 g of bovine serum albumin (BSA). Weigh 1.5 g of mannitol as a freeze-drying protectant. Weigh 0.2g of progesterone API and prepare appropriate amounts of dichloromethane and deionized water. Nanoparticle preparation mPEG-PCL, composite surfactant and progesterone were dissolved in 10 mL of dichloromethane to form an oil phase; BSA was dissolved in 30 mL of deionized water to form an aqueous phase. The oil phase was slowly added dropwise to the water phase at a stirring speed of 1200 rpm to form a primary emulsion. The primary emulsion was transferred to a probe sonicator and sonicated at a power of 250 W for 12 min to form a stable nanoemulsion. Freeze-drying Add 1.5 g of freeze-dried protective agent to the nanoemulsion and stir evenly. The nanoemulsion containing the lyoprotectant was dispensed into lyophilization bottles and pre-frozen at -40°C for 3.5 h. Vacuum freeze-drying was carried out. In the first stage, the temperature was increased from -40℃ to -20℃, the vacuum degree was maintained at 15Pa, and the time was 5.5h; in the second stage, the temperature was increased from -20℃ to 0℃, the vacuum degree was maintained at 8Pa, and the time was 9h; in the third stage, the temperature was increased from 0℃ to 20℃, the vacuum degree was maintained at 3Pa, and the time was 4.5h. Preparation molding The freeze-dried powder was reconstituted with saline to produce a progesterone protein nanoparticle sustained-release preparation. Testing showed that the initial average particle size of the nanoparticles in this preparation was 110 nm. After seven days of storage in saline at 37°C, the average particle size decreased to 300 nm. The activity retention rate of the protein carrier was 75%. By comparing Examples 1 and 2 with Comparative Example 1, it can be clearly seen that the mixture of trehalose and sorbitol used in the present invention as a lyoprotectant has significant advantages in inhibiting nanoparticle aggregation and protecting the activity of protein carriers.

[0017] The following are supplementary experimental designs and sample data for the three parts of complex surfactants, lyoprotectants, and nanoparticle stability mechanisms: 1. Verification of the effect of compound surfactants Experimental purpose: To verify the effect of Tween 80 / SDS (3:1) complex system on the structure of BSA.

[0018] Experimental design: Circular dichroism (CD) analysis: Control group: pure BSA solution (1 mg / mL in PBS).

[0019] Experimental group: BSA + complex surfactant (Tween 80 0.6 mg / mL + SDS 0.2 mg / mL).

[0020] Detection conditions: Far UV region (190-250 nm), 25°C.

[0021] Example data: | Group | α-helix content (%) | β-sheet content (%) | Random coil (%) | | Pure BSA | 55±2 | 20±1 | 25±1 | | BSA+complex | 53±3 | 21±2 | 26±2 | Conclusion: The compound surfactant did not significantly change the secondary structure of BSA (p>0.05), supporting its safety.

[0022] Supplementary experiments: SDS critical micelle concentration (CMC) verification: The actual CMC of SDS in the complex system was determined by the conductivity method (should be lower than 8 mM when used alone) to ensure that it did not form free micelles to destroy proteins.

[0023] 2. Lyoprotectant concentration optimization experiment Experimental purpose: To determine the optimal concentration of trehalose / sorbitol (2:1).

[0024] Experimental design: Set the lyoprotectant concentration gradient: 5%, 7.5%, 10% (w / v).

[0025] Evaluation indicators: Protein activity retention (HPLC determination).

[0026] Nanoparticle size after reconstitution (DLS).

[0027] Appearance of freeze-dried powder (whether it collapses or clumps).

[0028] Example data: | Concentration (%) | Protein activity retention (%) | Reconstituted particle size (nm) | Appearance | | 5 | 92±3 | 85±5 | Slight contraction | | 7.5 | 95±2 | 80±4 | Loose and porous | | 10 | 94±2 | 82±5 | Slightly sticky| Conclusion: 7.5% concentration has the best comprehensive performance and is recommended as the standard process parameter.

[0029] Supplementary experiments: Osmotic stress test: Measure the osmotic pressure of sorbitol solutions of varying concentrations (e.g., using a freezing point osmometer) to ensure that the concentration is ≤500 mOsm / kg (biocompatible range).

[0030] 3. Verification of Nanoparticle Stability Mechanism Experimental purpose: To clarify the contribution of steric hindrance and zeta potential of PEG to its stability in saline.

[0031] Experimental design: Salt ion stability experiment: The nanodispersions were placed in PBS with different NaCl concentrations (0.9%, 1.5%, and 2.0%) and incubated at 37°C for 24 h.

[0032] Detect particle size change (DLS) and zeta potential.

[0033] Example data: | NaCl concentration (%) | Initial particle size (nm) | Particle size after 24 hours (nm) | Zeta potential (mV) | | 0.9 | 80±5 | 85±6 | -28±2 | | 1.5 | 80±5 | 120±10 | -25±3 | | 2.0 | 80±5 | 250±20 (aggregation) | -18±4 | Conclusion: In normal saline (0.9% NaCl), the nanoparticles rely on PEG steric hindrance (rather than zeta potential alone) to maintain stability.

[0034] Supplementary experiments: PEG layer thickness characterization: Determine the PEG layer thickness (expected 5-10 nm) by X-ray photoelectron spectroscopy (XPS) or dynamic light scattering (DLS).

[0035] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for solubilizing and sustained-release of a progesterone protein nano-sustained-release preparation, characterized in that: The following steps are involved: Raw material preparation: including the preparation of skeleton materials, adsorption layer materials, protein carriers, and freeze-drying protective agents; Nanoparticle preparation: Drug-loaded nanoparticles were prepared by emulsification-ultrasound method; Freeze-drying treatment: using step-by-step temperature freeze-drying process; Preparation molding: The freeze-dried powder is reconstituted to form a stable nano-dispersion with an average particle size of ≤100nm.

2. The solubilization and sustained-release method of the progesterone protein nano sustained-release preparation according to claim 1, characterized in that: The raw material preparation includes the preparation of the skeleton material, the preparation of the adsorption layer, the preparation of the protein carrier, the preparation of the lyophilization protective agent and the preparation of other raw materials; 2.

1. Select amphiphilic polymers with a molecular weight of 5000-15000Da, such as methoxypolyethylene glycol-polycaprolactone, as the backbone material of the nanoparticles; 2.

2. A composite system of a nonionic surfactant, Tween 80, and an anionic surfactant, sodium lauryl sulfate, was selected. The mass ratio of Tween 80 to SDS was 3:

1. At this ratio, the surface zeta potential of the nanoparticles was measured by dynamic light scattering to be -30±2 mV, and an adsorption layer with a thickness of 5±1 nm was formed. The composite surfactant was able to form a dense adsorption layer on the surface of the nanoparticles. 2.

3. Bovine serum albumin is used as the protein carrier; 2.

4. Prepare a lyoprotectant. Use a mixture of trehalose and sorbitol in a mass ratio of 2:1 to replace traditional mannitol. Trehalose and sorbitol can form hydrogen bonds with protein molecules, effectively protecting the structure and function of the protein carrier during the lyophilization process and inhibiting its denaturation. 2.

5. Prepare progesterone API, deionized water and other raw materials.

3. The solubilization and sustained-release method of the progesterone protein nano sustained-release preparation according to claim 1, characterized in that: The nanoparticle preparation comprises dissolving a certain amount of amphiphilic polymer, compound surfactant and progesterone in an organic solvent, dichloromethane, to form an oil phase; dissolving a protein carrier in deionized water to form an aqueous phase, wherein the volume ratio of the oil phase to the aqueous phase is 1:3; Under a stirring speed of 1200±50rpm, the oil phase was added dropwise at a rate of 0.5mL / min to form a primary emulsion; The primary emulsion was transferred to a probe sonicator and sonicated for 12±1 min at a power of 250 W and a frequency of 20 kHz. The resulting nanoemulsion had a particle size distribution (PDI) of ≤0.

15.

4. The solubilization and sustained-release method of the progesterone protein nano sustained-release preparation according to claim 1, characterized in that: The freeze-drying process refers to adding a freeze-drying protectant to the nanoemulsion at a mass concentration of 5-10%, and packaging the nanoemulsion containing the freeze-drying protectant into freeze-drying bottles. The pre-freezing temperature is -40°C and the pre-freezing time is 3-4 hours to completely freeze the nanoemulsion and perform vacuum freeze-drying. The freeze-drying process is divided into three stages: In the first stage, the temperature is raised from -40℃ to -20℃, and the vacuum degree is maintained at 10-20Pa for 5-6h; In the second stage, the temperature is raised from -20℃ to 0℃, and the vacuum degree is maintained at 5-10Pa for 8-10h; In the third stage, the temperature is raised from 0°C to 20°C, and the vacuum degree is maintained at 2-5 Pa for 4-5 hours; The specific surface area of ​​the lyophilized nanoparticles was determined to be 35±3m² / g by the BET method, and the protein activity retention rate after reconstitution of the native conformation ratio was determined by HPLC to be ≥95%.

5. The solubilization and sustained-release method of the progesterone protein nano sustained-release preparation according to claim 1, characterized in that: The freeze-dried powder was added to normal saline at a ratio of 1:10 and subjected to 30-second, 40kHz ultrasonic treatment. The following results were obtained: Dynamic light scattering showed an average particle size of 80 ± 5 nm; Transmission electron microscopy showed that the core-shell structure was intact; The particle size change rate is less than 5% under 4°C storage conditions within 3 months; The in vitro release curve of the reconstituted preparation conforms to the Higuchi equation, and the cumulative release rate in pH 7.4 phosphate buffer is 65±5% over 24 hours and reaches more than 90% over 72 hours.