An (s)-equol liposome composition and a method for preparing the same

Abstract

This invention discloses a (S)-equorol liposome composition and its preparation method, belonging to the field of liposome preparation technology. This invention combines microfluidic technology with solvent-free microfluidic technology for the preparation of (S)-equorol liposomes. The synergistic effect of these two technologies allows for precise control of the average particle size of the resulting liposomes between 50 and 200 nm, with a PDI < 0.2 and an encapsulation efficiency ≥ 85%. The solvent-free process completely eliminates the risk of residual organic solvents. In vivo experiments demonstrate that the oral bioavailability of the product of this invention is 2.8 times higher than that of free (S)-equorol, and the Tmax is extended from 1.5 h to 4.5 h, truly achieving the dual goals of solubilization and controlled release. Furthermore, the powder formulation retains 2.75 times the relative bioavailability after reconstitution, exhibiting good stability and practicality. This composition can be formulated into a liquid product for direct use or spray-dried or freeze-dried into a powder product for long-term storage, showing broad application prospects in pharmaceuticals, health foods, dietary supplements, and functional foods.

Description

Technical Field

[0001] This invention belongs to the field of liposome preparation technology, and specifically relates to a (S)-estrol liposome composition and its preparation method. Background Technology

[0002] (S)-Estradiol is an isoflavone metabolite produced from daidzein by intestinal flora. It possesses selective estrogen receptor modulator activity and shows broad application prospects in alleviating menopausal syndrome, preventing osteoporosis, and improving skin aging. However, (S)-Estradiol has poor water solubility, with a solubility of only about 10-20 μg / mL in water, making it difficult to prepare high-concentration liquid formulations. Furthermore, after oral administration, (S)-Estradiol easily crystallizes in the aqueous environment of the gastrointestinal tract and is susceptible to enzymatic degradation and the first-pass effect, directly impacting its bioavailability. In addition, significant metabolic differences exist among individuals, severely limiting its application in health supplements and pharmaceuticals.

[0003] Microfluidics and microfluidics are two representative technologies explored in recent years for liposome preparation. Microfluidics can precisely control the mixing of two-phase fluids in micrometer-scale channels to achieve the self-assembly of lipid molecules, offering advantages such as controllable particle size and uniform distribution. However, most existing microfluidic methods still rely on organic solvents as the dissolving medium for lipid materials, making it difficult to completely eliminate the risk of solvent residue. More importantly, due to the limitations of microchannel size, the throughput of single-channel microfluidic chips is extremely low, and even using multi-channel parallel chips is difficult to meet the needs of large-scale industrial production. Solvent-free microfluidics utilizes high-pressure shear force to directly self-assemble phospholipids into liposomes in an aqueous phase, offering advantages such as no solvent residue, continuous production capability, and high throughput, theoretically suitable for industrial scale-up. However, the applicant found that liposomes prepared using microfluidic technology alone have a wide particle size distribution (PDI usually > 0.3) and low encapsulation efficiency (usually < 75%) because the formation of liposomes during high-pressure shearing is random and uncontrollable. Furthermore, the batch-to-batch reproducibility is poor, making it difficult to meet the stringent requirements for product quality uniformity in the food, health products, and pharmaceutical fields.

[0004] Therefore, a single technical approach cannot simultaneously achieve high encapsulation efficiency, narrow particle size distribution, good batch reproducibility, and industrial-scale production of liposomes without using any organic solvents. Summary of the Invention

[0005] [Technical Issues] This invention provides a method for preparing (S)-equol liposomes that is solvent-free, process-controllable, and suitable for industrial scale-up, achieving multiple objectives such as improving (S)-equol solubility, prolonging (S)-equol retention time in vivo, and enhancing (S)-equol oral bioavailability.

[0006] [Technical Solution] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] The core design concept of this invention is to address the inherent defects of (S)-equorol, such as low solubility and poor absorption. It adopts a technical strategy that combines solubilization and controlled release. Through the synergistic effect of precise microfluidic self-assembly and high-pressure microfluidic homogenization, it achieves efficient nano-encapsulation of (S)-equorol under completely solvent-free conditions. At the same time, it precisely controls the liposome particle size within the optimal absorption range, thereby achieving multiple goals such as improving solubility, prolonging in vivo retention time, and enhancing oral bioavailability.

[0009] The technical solutions involved in this invention include: First, the present invention provides a method for preparing a (S)-equadol liposome composition, comprising the following steps: (1) Premixing and dispersing: Phospholipids, (S)-estrol, stabilizers and antioxidants are added to water and dispersed evenly to form a crude liposome suspension; (2) Initial mixing in microfluidic chip: The crude liposome suspension obtained in step (1) is introduced into the inlet channel of the microfluidic chip. Through the fluid focusing and passive mixing mechanism in the microchannel, an initial nanoliposome suspension with uniform particle size is formed. (3) Solvent-free microfluidic homogenization: The initial nanoliposome suspension obtained in step (2) is subjected to high-pressure microfluidic homogenization. The homogenization pressure is 10,000~60,000 psi, and the homogenization cycle is 3~10 times to obtain a (S)-estrol liquid liposome composition with uniform particle size. The (S)-estrol liquid liposome composition obtained in step (3) may or may not be dried; if dried, a (S)-estrol liposome composition powder product is obtained.

[0010] In one embodiment of the present invention, in step (1), the crude liposome suspension comprises, by mass percentage: 1.0%~15.0% phospholipids, 0.1%~5.0% (S)-estradiol, 0~5.0% stabilizer, 0.05%~1.0% antioxidant, and water to the remainder up to 100%.

[0011] In one embodiment of the present invention, in step (1), the phospholipid is selected from one or more of soybean lecithin, sunflower lecithin, and egg yolk lecithin; the phospholipid contains 30% to 90% phosphatidylcholine (PC).

[0012] In one embodiment of the present invention, in step (1), the stabilizer is selected from one or more of cholesterol, bile salts (such as sodium deoxycholate), and polyols (such as sorbitol and xylitol). In one embodiment of the present invention, in step (1), the antioxidant is selected from one or more of vitamin E, ascorbyl palmitate, and rosemary extract.

[0013] In one embodiment of the present invention, in step (1), the crude liposome suspension further includes the addition of 0-10.0% lyophilization protectant; the lyophilization protectant is selected from one or more of trehalose, sucrose, lactose, maltose, mannitol, sorbitol and xylitol.

[0014] In one embodiment of the present invention, when the composition is stored in powder form for a long period of time, the amount of lyophilization protectant added is 1.0% to 10.0%.

[0015] In one embodiment of the present invention, in step (1), the condition for uniform dispersion is to stir and disperse at 40~60°C for 10~30 min.

[0016] In one embodiment of the present invention, in step (2), the flow rate of the crude liposome suspension into the inlet channel of the microfluidic chip is 0.5 mL / min to 5 mL / min.

[0017] In one embodiment of the present invention, in step (2), the hydraulic diameter of the microchannel of the microfluidic chip is 50~200 μm.

[0018] In one embodiment of the present invention, in step (2), the microfluidic chip is a multilayer parallel microchannel chip, which includes at least two inlet channels and one outlet channel. The inlet channels flow into the mixing channel after passing through the fluid focusing area. The ratio of the length of the mixing channel to the hydraulic diameter is 10:1 to 100:1 to obtain a sufficient fluid mixing effect.

[0019] In one embodiment of the present invention, in step (3), the homogenization pressure is preferably 20,000 to 40,000 psi.

[0020] In one embodiment of the present invention, in step (3), the temperature condition for high-pressure microfluidic homogenization is 4~25°C.

[0021] In one embodiment of the present invention, in step (3), the homogenization cycle is preferably performed 4 to 8 times.

[0022] In one embodiment of the present invention, the method further includes: filling the liposome composition obtained in step (3) into capsules or sterile bottles to obtain a liquid product, or further drying it to obtain a powder product.

[0023] In one embodiment of the present invention, the drying process further includes adding a freeze-drying protectant to the liquid liposome composition before drying, followed by drying to obtain a powder product.

[0024] In one embodiment of the present invention, the amount of the lyophilization protectant relative to the liquid liposome composition is 1.0 wt% to 10.0 wt%.

[0025] In this invention, the powder product can be prepared using two methods of adding the freeze-drying protectant: Method 1 is a one-step method, in which the freeze-drying protectant is simultaneously added to water and dispersed during the premixing and dispersion stage in step (1); Method 2 is a two-step method, in which the freeze-drying protectant is not added in step (1), a liquid liposome composition is first prepared, and then the freeze-drying protectant is mixed evenly with the liquid liposome before spray drying. Both methods can yield powder products with similar performance.

[0026] In one embodiment of the present invention, the drying process is selected from spray drying or freeze drying. Specifically, the inlet air temperature of spray drying is 120~180℃, and the outlet air temperature is 60~90℃. For freeze drying, the pre-freezing temperature is -40~-20℃, the primary drying temperature is -20~0℃, the secondary drying temperature is 20~30℃, and the vacuum degree is ≤20 Pa.

[0027] The present invention provides a (S)-estrol liposome composition based on the above preparation method.

[0028] In one embodiment of the present invention, when the (S)-estrol liposome composition is (S)-estrol liquid liposome, its average particle size is 50~200 nm, polydispersity index (PDI) < 0.2, and encapsulation efficiency ≥ 85%.

[0029] In one embodiment of the present invention, when the (S)-estrol liposome composition is a powder product, after the powder product is redissolved in water, the average particle size of the liposomes increases by no more than 20%, the polydispersity index (PDI) is <0.3, and the encapsulation retention rate is ≥85%.

[0030] The present invention also provides the application of the above-mentioned (S)-estrol liposome composition in the fields of health foods, dietary supplements and functional foods.

[0031] The present invention also provides the use of the above-mentioned (S)-estrol liposome composition in the preparation of health foods or medicines for improving skin aging, relieving menopausal syndrome, and preventing osteoporosis.

[0032] [Beneficial Effects] This invention addresses the two inherent drawbacks of (S)-equorol: poor water solubility and low oral bioavailability. It innovatively combines the solubilizing effect of nanoliposomes with controlled-release properties achieved through precise particle size control. On one hand, the hydrophobic (S)-equorol is encapsulated within a lipid membrane by the phospholipid bilayer structure of the liposomes, significantly improving its solubility and dispersion stability in aqueous environments. On the other hand, by precisely controlling the liposome particle size within the optimal range of 50–200 nm, it can be effectively absorbed through intestinal epithelial cells while avoiding excessively rapid drug release, achieving a long-lasting sustained-release effect. Furthermore, this invention eliminates the use of organic solvents in traditional liposome preparation. Phospholipids and (S)-equorol are directly dispersed in the aqueous phase, and self-assembly is completed through microfluidic initial mixing and microfluidic homogenization, eliminating any risk of organic solvent residue and fully complying with regulatory requirements in the food and health product field. Through the precise fluid control of the microfluidic chip and the high-energy synergistic effect of the homogenization of the microjets, the average particle size of the prepared (S)-estradiol liposomes can be controlled between 50 and 200 nm, the polydispersity index (PDI) is <0.2, and the encapsulation efficiency can reach more than 85%, which is far superior to liposomes prepared by traditional methods.

[0033] Furthermore, the liposome composition prepared using the method of this invention shows no significant change in particle size and encapsulation efficiency after storage at 4°C or 25°C for more than 6 months. The powder composition also exhibits high structural stability during storage and reconstitution, enabling long-term preservation. After reconstitution, it retains the intact liposome structure and its high encapsulation efficiency and bioavailability. This composition can be widely used in pharmaceuticals, health foods, dietary supplements, and functional foods. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 The dynamic light scattering (DLS) particle size distribution of the (S)-estrol liquid liposomes prepared in Example 1 of this invention shows an average particle size of approximately 118 nm and a PDI of 0.15.

[0035] Figure 2 The DLS particle size distribution diagram of the (S)-estradiol liposome powder prepared in Example 6 of this invention after rehydration shows an average particle size of about 126 nm and a PDI of 0.18.

[0036] Figure 3 This is a comparison of the blood concentration-time curves of (S)-estrol liposomes prepared in Example 1 of the present invention and free (S)-estrol after oral administration in rats.

[0037] Figure 4 The diagram shows a comparison of liposome particle size distributions prepared using different processes in Example 1 and Comparative Examples 1-3 of the present invention. The liposomes include: liquid liposome product of Example 1 of the present invention, powder product of Example 1 of the present invention after rehydration, liquid liposome product of Comparative Example 1 (microfluidic homogenization only), liquid liposome product of Comparative Example 2 (microfluidic only), and liquid liposome product of Comparative Example 3 using the traditional thin-film hydration method. Detailed Implementation

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0039] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0040] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0041] Unless otherwise specified, the raw materials and equipment used in the embodiments of this invention can be obtained commercially: sunflower lecithin (70% PC content, ADM, USA); soybean lecithin (50% PC content, Lipoid, Germany); (S)-estrol (purity ≥99%, Xianchi Biopharmaceutical (Hangzhou Qiandao Lake) Co., Ltd., CellAuto+). ® ); Sodium deoxycholate (Sinopharm Chemical Reagent Co., Ltd.).

[0042] The rehydration involved in this invention specifically refers to the process of adding powdered liposome products to deionized water at a solid-liquid ratio of 1:10 (g / mL), and gently shaking or stirring to redisperse them to form a liposome suspension, which is used to evaluate the integrity of the liposome structure and the retention of performance after the powdered product is reconstituted.

[0043] Example 1: Preparation of (S)-Estrol Liquid Liposome Composition Raw material formula (per 100g): (S)-Estrol 0.5g, Sunflower lecithin (PC≥70%) 8.0g, Sodium deoxycholate 1.0g, Vitamin E 0.1g, Deionized water 90.4g.

[0044] Preparation steps: (1) Premixing and dispersing: Weigh sunflower lecithin, sodium deoxycholate and vitamin E according to the ratio, add them to 80 g of deionized water, heat to 50±2℃, and stir magnetically at 300 rpm for 15 min until a uniform milky white suspension is formed; slowly add (S)-estradiol powder to the above suspension, keep at 50℃ and continue stirring for 5 min to obtain crude liposome suspension; (2) Microfluidic pre-homogenization: The crude liposome suspension was introduced into the microfluidic chip (microchannel hydraulic diameter 100 μm, mixing channel length-to-diameter ratio 30:1) at a constant flow rate of 2.0 mL / min. The chip was placed in a constant temperature water bath at 15℃, and the initial nanoliposome suspension at the outlet was collected. (3) Microfluidic homogenization: The initial suspension was transferred to a high-pressure microfluidic homogenizer, the feed temperature was controlled at 15°C, and the homogenization was carried out 6 times under a pressure of 30,000 psi to obtain a (S)-estrol liquid liposome composition with uniform particle size. (4) Volumetric filling: Add deionized water to 100 g, filter through a 0.22 μm filter membrane for sterilization, fill into brown vials, seal with nitrogen, and store at 4°C.

[0045] Alternatively, the liquid liposome composition obtained in step (3) can be spray-dried to obtain the corresponding powder product (5.0 wt% trehalose is added as a protective agent, and the inlet air temperature is controlled at 140°C, the outlet air temperature at 70°C, the feed flow rate at 10 mL / min, and the atomization pressure at 0.3 MPa).

[0046] Test results Liquid liposome composition product: average particle size 118.3±5.2 nm, PDI 0.15±0.02, Zeta potential -38.6±2.1 mV, encapsulation efficiency 92.4±1.8%, ethanol residue not detected (<1 ppm).

[0047] Example 2: Preparation of (S)-Estrol Liposome Composition Powder Product Raw material formula (per 100g): (S)-estradiol 2.0 g, soybean lecithin (PC≥50%) 12.0 g, cholesterol 2.0 g, rosemary extract 0.2 g, sorbitol 3.0 g, deionized water 70.8 g.

[0048] Preparation steps (1)-(3) are the same as in Example 1, to obtain a (S)-estrol liquid liposome composition with uniform particle size; then spray drying is performed (inlet air temperature 150℃, outlet air temperature 80℃, feed rate 5 mL / min) to obtain a light yellow powder.

[0049] Test results The liquid liposome composition had an average particle size of 145.7±6.8 nm, a PDI of 0.17±0.03, and an encapsulation efficiency of 88.7±2.1%.

[0050] Example 3 Referring to Example 1, the flow rate in step (2) and the homogenization pressure in step (3) were adjusted, while other steps remained unchanged, to obtain the corresponding liquid liposome composition product. As shown in Table 1: Table 1

[0051] The test results of the obtained liposome composition products are shown in Table 2.

[0052] Table 2

[0053] The results show that, within the parameter range required by this invention, high-quality products with PDI < 0.2 and encapsulation efficiency > 85% can be obtained.

[0054] Example 4: Oral bioavailability test in rats SPF-grade male SD rats (n=6 / group) were administered the drug via gavage at a dose of 50 mg / kg (calculated as (S)-equadol). Blood drug concentrations were determined by LC-MS / MS, and the results are shown in Table 3.

[0055] Table 3

[0056] The results showed that the oral bioavailability of the liposome group was increased by 2.80 times, and the in vivo retention time was significantly prolonged.

[0057] Example 5: Correlation experiment between preparation parameters and bioavailability To quantitatively reveal the effects of the preparation process parameters (microfluidic flow rate, microfluidic homogenization pressure, and homogenization cycle number) of the present invention on the structural parameters (particle size, PDI, encapsulation efficiency) and in vivo oral bioavailability of liposomes, multiple parameter gradients were set up. Using the same basic formulation as in Example 1 ((S)-estrol 0.5 g, sunflower lecithin 8.0 g, sodium deoxycholate 1.0 g, vitamin E 0.1 g, deionized water to 100 g), the particle size, PDI, and encapsulation efficiency of each group of liposomes were measured, and representative groups were selected for in vivo pharmacokinetic studies in rats.

[0058] 5.1 Effects of microfluidic flow rate on liposome structure and bioavailability The microfluidic homogenization pressure was fixed at 30,000 psi, and the number of cycles was 6. The microfluidic flow rates were set to 0.5, 1.0, 2.0, 3.0, and 5.0 mL / min, respectively. Other steps were the same as in Example 1, and 5 groups of liposome samples (numbered F1 to F5) were prepared. The measurement results are shown in Table 4.

[0059] Table 4. Structural parameters of liposomes at different microfluidic flow rates

[0060] Three groups, F1 (flow rate 0.5 mL / min), F3 (flow rate 2.0 mL / min), and F5 (flow rate 5.0 mL / min), were selected for in vivo pharmacokinetic experiments in rats. Free (S)-estrol was used as a control. The relative bioavailability was calculated, and the results are shown in Table 5.

[0061] Table 5. In vivo pharmacokinetic parameters of samples with different microfluidic flow rates

[0062] Results analysis: When the microfluidic flow rate was 2.0 mL / min, the liposomes exhibited the lowest PDI (0.15), the highest encapsulation efficiency (92.8%), and both the in vivo AUC and Cmax reached their maximum values. At excessively low flow rates (0.5 mL / min), prolonged mixing time led to partial liposome fusion, increasing the PDI, decreasing the encapsulation efficiency, and reducing bioavailability. At excessively high flow rates (5.0 mL / min), insufficient mixing resulted in smaller liposome particle sizes but a wider distribution, increased drug leakage, and decreased in vivo absorption.

[0063] Conclusion: The optimal flow rate is 2.0 mL / min. At this parameter, the liposome structure is the most uniform and the in vivo absorption effect is the best.

[0064] 5.2 Effect of microfluidic homogenization pressure on liposome structure and bioavailability A fixed microfluidic flow rate of 2.0 mL / min and 6 cycles were performed. Homogenization pressures of 15000, 20000, 25000, 30000, 35000, 40000, and 45000 psi were set to prepare 7 groups of liposome samples (numbered P1 to P7). The results are shown in Table 6.

[0065] Table 6. Structural parameters of liposomes under different microjet pressures

[0066] Three groups, P2 (20000 psi), P4 (30000 psi), and P6 (40000 psi), were selected for in vivo pharmacokinetic experiments in rats. The results are shown in Table 7.

[0067] Table 7. In vivo pharmacokinetic parameters of samples with different microjets of pressure

[0068] Results analysis: At excessively low pressure (20,000 psi), the liposome particle size was too large (168.4 nm), the encapsulation efficiency was low (87.2%), and some multilayer vesicles were not completely broken, resulting in slow but incomplete absorption in vivo. At excessively high pressure (40,000 psi), although the particle size was small (92.7 nm), the encapsulation efficiency decreased (86.3%), and the PDI increased, indicating that some liposomes ruptured or the drug leaked. The optimal pressure was 30,000 psi, at which point the liposome particle size was approximately 118 nm, which is within the optimal size range for intestinal lymphatic transport, resulting in the highest encapsulation efficiency (92.8%) and the largest in vivo AUC.

[0069] 5.3 Effect of homogenization cycle number on liposome structure and bioavailability Five groups of liposome samples (numbered C1 to C5) were prepared with a fixed microfluidic flow rate of 2.0 mL / min, a homogenization pressure of 30,000 psi, and 2, 4, 6, 8, and 10 cycles. The results are shown in Table 8.

[0070] Table 8. Structural parameters of liposomes after different homogenization cycles

[0071] Three groups, C2 (4 times), C3 (6 times), and C4 (8 times), were selected for in vivo pharmacokinetic experiments in rats. The results are shown in Table 9.

[0072] Table 9. In vivo pharmacokinetic parameters of samples with different homogenization cycles

[0073] Results analysis: When the number of cycles was insufficient (4 cycles), the liposome particle size was too large, the PDI was too high, and the encapsulation efficiency was insufficient, resulting in incomplete absorption in vivo. When the number of cycles was too high (8 cycles or more), although the particle size was further reduced, repeated high-pressure shearing may have damaged the liposome membrane and caused drug leakage, leading to a decrease in encapsulation efficiency and a decrease in in vivo AUC. The optimal number of cycles was 6, at which point the liposome structure was intact, the encapsulation efficiency was high, and the in vivo absorption effect was the best.

[0074] 5.4 Multi-parameter collaborative optimization and in vivo bioavailability prediction model Based on the above single-factor experimental data, a multi-parameter regression model was established using the response surface methodology, with AUC0-24h as the response variable. The following quantitative relationship was obtained (goodness of fit R² = 0.96): AUC0-24h (ng·h / mL) = -3850 + 12.5×A + 0.18×B + 85×C - 0.0025×A×B - 0.35×A×C - 0.008×B×C Where A is the microfluidic flow rate (mL / min), B is the microjet pressure (psi), and C is the number of cycles.

[0075] The optimal parameter combination predicted by the model was: microfluidic flow rate 2.1 mL / min, microjet pressure 30500 psi, and 6.2 cycles, with a predicted AUC0-24h of 2480 ng·h / mL. This result is in high agreement with the measured value in Example 1 (2456.8 ng·h / mL), verifying the reliability of the model.

[0076] Conclusion: The preparation parameters involved in the method of this invention (microfluidic flow rate 0.5~5 mL / min, microjet pressure 20000~40000 psi, 4~8 cycles) can stably obtain (S)-estrol liposomes with high encapsulation efficiency (≥85%), small particle size (50~200 nm), and narrow distribution (PDI<0.2). Among them, the optimal parameter combination (flow rate 2.0 mL / min, pressure 30000 psi, 6 cycles) can achieve an oral bioavailability of 2.80 times that of free (S)-estrol, and when the parameters fluctuate within the optimal window, the bioavailability can still be maintained at more than 2.5 times that of free drug, demonstrating the robustness and superiority of the process of this invention.

[0077] Example 6: Preparation of (S)-Estradiol Liposome Powder Product by Spray Drying Raw material formulation (100g liposome suspension): 100g of liquid liposomes prepared in Example 1 were used, and 5.0g of trehalose was added as a spray drying protectant.

[0078] Preparation steps: 1) Addition of protective agent: Add 5 wt% trehalose to the liquid liposomes prepared in Example 1 and stir at 300 rpm for 10 min until completely dissolved; 2) Spray drying: The mixture is fed into a spray dryer, with the inlet air temperature set to 140℃, the outlet air temperature to 70℃, the feed flow rate to 10 mL / min, and the atomization pressure to 0.3 MPa. The dried liposome powder is then collected. 3) Packaging: Seal the powder in an aluminum foil bag and store at room temperature away from light.

[0079] Test results: Powder properties: Off-white loose powder with good flowability and an angle of repose of <35°; Moisture content: 2.8±0.3%; Rehydration performance: It can be completely dissolved by adding deionized water at a solid-liquid ratio of 1:10 and shaking for 30 s. After rehydration, the average particle size is 126.5±6.1 nm, PDI is 0.18±0.02, encapsulation efficiency is 87.2±1.9%, and encapsulation retention rate is 94.4%.

[0080] Example 7: Preparation of (S)-Estrol Liposome Powder Product by Freeze-Drying Raw material formulation (100g liposome suspension): 100g of liquid liposomes prepared in Example 1 were used, and 4.0g of mannitol was added as a freeze-drying protectant.

[0081] Preparation steps: 1) Addition of protective agent: Add mannitol to the liquid liposomes prepared in Example 1, stir at 300 rpm for 10 min until completely dissolved, and dispense into vials; 2) Pre-freezing: Place the vials in a freeze dryer and pre-freeze at -45℃ for 6 hours; 3) Sublimation drying: Set the sublimation pressure to 20 Pa, the sublimation temperature to -20℃, and the sublimation time to 18 h; 4) Desorption and drying: Set the desorption temperature to 25℃ and the desorption time to 3 hours; 5) Plugging and sealing: After freeze-drying, fill with nitrogen and plug, seal with aluminum cap, and store at room temperature away from light.

[0082] Test results: Powder properties: White, loose cake-like powder that disperses easily with light shaking and has good rehydration properties; Moisture content: 1.5±0.2%; Rehydration performance: When deionized water is added at a solid-liquid ratio of 1:10 and shaken for 10 seconds, it can be completely dissolved. After rehydration, the average particle size is 122.4±5.8 nm, PDI is 0.17±0.02, encapsulation efficiency is 89.5±1.7%, and encapsulation retention rate is 96.9%.

[0083] Example 8 Stability Study 8.1 Stability of liquid dosage forms The samples from Example 1 were stored at 4°C and 25°C, respectively, in the dark for 6 months.

[0084] At 4℃, after 6 months, the particle size increased from 118.3 nm to 125.6 nm (change rate +6.2%), and the encapsulation efficiency decreased from 92.4% to 88.7% (retention rate 96.0%). At 25℃, after 6 months, the particle size increased to 138.9 nm (change rate +17.4%), and the encapsulation efficiency decreased to 84.2% (retention rate 91.1%). No visible precipitation or stratification was observed at any time point.

[0085] 8.2 Stability of Powder Formulation The powder samples from Example 6 (spray drying) and Example 7 (freeze drying) were stored at 25°C in the dark for 12 months.

[0086] Spray-dried powder: After 12 months of rehydration, the particle size was 135.2 nm (change rate +6.9%), and the encapsulation efficiency was 82.6% (retention rate 94.7%). Freeze-dried powder: After 12 months of rehydration, the particle size was 128.7 nm (change rate +5.1%), and the encapsulation efficiency was 85.3% (retention rate 95.3%). Neither group of powders showed any signs of moisture absorption or clumping, and their rehydration performance did not decrease significantly.

[0087] Comparative Example 1 Referring to Example 1, step (2) is omitted, and the crude liposome suspension obtained in step (1) is directly subjected to high-pressure microfluidic homogenization treatment, with other aspects remaining unchanged, to obtain the corresponding liquid liposome product.

[0088] Powder product preparation: Take 100g of the liquid liposomes obtained in Comparative Example 1, add 5.0g of trehalose as a freeze-drying protectant, stir at 300rpm for 10min until completely dissolved, and prepare the powder product using the same spray drying process as in Example 6 (inlet air temperature 140℃, outlet air temperature 70℃, feed flow rate 10mL / min, atomization pressure 0.3MPa).

[0089] Comparative Example 2 Referring to Example 1, step (3) is omitted, and the initial nanoliposome suspension obtained in step (2) is directly bottled to a fixed volume to obtain the corresponding liquid liposome product.

[0090] Powder product preparation: Take 100g of the liquid liposomes obtained in Comparative Example 2, add 4.0g of mannitol as a freeze-drying protectant, stir at 300rpm for 10min until completely dissolved, and prepare the powder product using the same freeze-drying process as in Example 7 (pre-freezing at -45℃ for 6h, sublimation pressure 20Pa, sublimation temperature -20℃, sublimation time 18h, desorption temperature 25℃, desorption time 3h).

[0091] Comparative Example 3 Liquid products prepared by the traditional thin-film hydration method-ethanol solvent method (this method was first proposed by Bangham et al. in 1965, see Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of Molecular Biology, 1965, 13(1): 238-252; it is still widely used in liposome preparation in recent years, see Chen Weijun, Qin Dongmei, Meng Lingyu, et al. Optimization of preparation process and quality evaluation of isovalerylshikonin liposomes. Chinese Pharmaceutical Journal, 2024, 59(19): 1843-1852; and Carroll J, Daly N, Mondala JRM, et al. Liposome synthesis protocol: synthesis of anionic and cationic unilamellar liposome nanoparticles using a thin film dispersed hydration and extrusion method. 2023.). Specific preparation steps: Weigh 12.0 g of soybean lecithin, 2.0 g of cholesterol, 2.0 g of (S)-estradiol, and 0.2 g of vitamin E, dissolve them in 200 mL of anhydrous ethanol, transfer them to a rotary evaporator flask, and remove the ethanol by rotary evaporation under reduced pressure in a water bath at 45 °C, forming a uniform lipid film on the inner wall of the flask; add 100 mL of PBS buffer at pH 7.4, hydrate at 45 °C for 30 min, and obtain a crude liposome suspension; sonicate with a probe for 10 min (power 300 W, 3 s operation followed by 2 s pause), filter through a 0.22 μm filter membrane to obtain the liquid liposome product.

[0092] Preparation of powdered products: Take 100 g of the liquid liposomes obtained in Comparative Example 3, add 5.0 g of sucrose as a freeze-drying protectant, stir at 300 rpm for 10 min until completely dissolved, and prepare powdered products by spray drying (inlet air temperature 140℃, outlet air temperature 70℃, feed flow rate 10 mL / min, atomization pressure 0.3 MPa).

[0093] The performance results of the liquid and powder samples of Example 1 and Comparative Examples 1-3 are shown in Table 10.

[0094] Table 10

[0095] The results showed that, compared with the control groups, the particle size, PDI, and encapsulation efficiency of the experimental group were significantly different after rehydration, whether in liquid or powder form. Microfluidic pre-dispersion dispersed large particles into the 200-300 nm range, providing a more uniform feed particle size distribution for microfluidics. Microfluidics further precisely controlled the self-assembly process, ultimately achieving narrow-distribution liposomes of ~100-120 nm. This demonstrates that the synergistic process of microfluidics and microfluidics not only improved the performance of liquid liposomes but also provided a stable foundation for the powdering process, significantly improving the performance retention rate after powder rehydration. The liposomes prepared by the combined process of this invention exhibited superior structural stability after freeze-drying, and the changes in particle size and encapsulation efficiency after rehydration were significantly smaller than those in the single-process control group.

[0096] Example 9: In vivo bioavailability verification experiment SPF-grade male SD rats (n=6 / group) were administered the drug via gavage at a dose of 50 mg / kg (calculated as (S)-equadol). Blood drug concentrations were determined by LC-MS / MS, and the results are shown in Table 11.

[0097] Table 11

[0098] The results showed that the (S)-estradiol liposomes prepared by the combined process of the present invention (microfluidic jet + microfluidic) had the highest bioavailability (280.3%), which was significantly better than that of the single process comparison example 1 (162.7%), comparison example 2 (149.7%) and the traditional process comparison example 3 (135.2%).

[0099] It is noteworthy that the liposomes of this invention maintained a relative bioavailability of 274.8% after freeze-drying, with a loss rate of only 2.0%, while the loss rates of the control groups were all around 6%. This result indicates that the combined process not only improved the initial encapsulation efficiency and particle size uniformity of the liposomes, but also significantly enhanced their structural stability, making them less prone to fusion or leakage during powdering.

[0100] Synergistic effect analysis showed that the relative bioavailability of the liquid liposome group in Comparative Example 1 was increased by 62.7% relative to free equol; the relative bioavailability of the liquid liposome group in Comparative Example 2 was increased by 49.7% relative to free equol; and the relative bioavailability of the liquid liposome group in Example 1 was increased by 180.3% relative to free equol, which was 32.0% higher than the combined effect of Comparative Example 1 and Comparative Example 2. This means that the bioavailability of the combined process is now better than the sum of the effects of the two steps alone, confirming the cascade optimization effect between microfluidic pre-fragmentation and microfluidic precise self-assembly.

[0101] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing a (S)-estrol liposome composition, characterized in that, Includes the following steps: (1) Premixing and dispersing: Phospholipids, (S)-estrol, stabilizers and antioxidants are added to water and dispersed evenly to form a crude liposome suspension; (2) Initial mixing in microfluidic chip: The crude liposome suspension obtained in step (1) is introduced into the inlet channel of the microfluidic chip. Through the fluid focusing and passive mixing mechanism in the microchannel, an initial nanoliposome suspension with uniform particle size is formed. (3) Solvent-free microfluidic homogenization: The initial nanoliposome suspension obtained in step (2) is subjected to high-pressure microfluidic homogenization. The homogenization pressure is 10,000~60,000 psi, and the homogenization cycle is 3~10 times to obtain a (S)-estrol liquid liposome composition with uniform particle size. The (S)-estrol liquid liposome composition obtained in step (3) may or may not be dried; if dried, a (S)-estrol liposome composition powder product is obtained.

2. The preparation method according to claim 1, characterized in that, In step (1), the crude liposome suspension comprises, by mass percentage: 1.0%~15.0% phospholipids, 0.1%~5.0% (S)-estradiol, 0~5.0% stabilizer, 0.05%~1.0% antioxidant, 0~10.0% lyophilization protectant, and water to the remainder up to 100%.

3. The preparation method according to claim 1, characterized in that, In step (1), the phospholipid is selected from one or more of soybean lecithin, sunflower lecithin, and egg yolk lecithin; the phospholipid contains 30% to 90% phosphatidylcholine.

4. The preparation method according to claim 1, characterized in that, In step (1), the stabilizer is selected from one or more of cholesterol, bile salts, and polyols; the antioxidant is selected from one or more of vitamin E, ascorbate palmitate, and rosemary extract; and the freeze-drying protectant is selected from one or more of trehalose, sucrose, lactose, maltose, mannitol, sorbitol, and xylitol.

5. The preparation method according to claim 1, characterized in that, In step (2), the flow rate of the crude liposome suspension into the inlet channel of the microfluidic chip is 0.5 mL / min to 5 mL / min; the hydraulic diameter of the microchannel of the microfluidic chip is 50 to 200 μm.

6. The preparation method according to claim 1, characterized in that, In step (3), the homogenization pressure is preferably 20,000 to 40,000 psi; the temperature conditions for high-pressure microjet homogenization are 4 to 25°C; and the homogenization cycle is 4 to 8 times.

7. The preparation method according to any one of claims 1-6, characterized in that, The method further includes: adding 0-10.0 wt% lyophilization protectant to the crude liposome suspension in step (1); or, Before drying, the liquid liposome composition is mixed with a lyophilization protectant and then dried to obtain a powder product; the amount of the lyophilization protectant relative to the liquid liposome composition is 1.0 wt% to 10.0 wt%.

8. A (S)-estrol liposome composition prepared by the preparation method according to any one of claims 1-7.

9. The use of the (S)-estrol liposome composition according to claim 8 in the fields of health foods, dietary supplements and functional foods.

10. Use of the (S)-estradiol liposome composition of claim 8 in the preparation of health foods or pharmaceuticals for improving skin aging, relieving menopausal syndrome, and preventing osteoporosis.

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