A doxorubicin hydrochloride liposome and its preparation method

Abstract

This invention belongs to the field of liposome formulation and preparation technology, specifically relating to a doxorubicin hydrochloride liposome and its preparation method. A specific concentration of ammonium sulfate aqueous solution and an oil phase solution containing lipid components are prepared. Microfluidic technology is used to mix the oil phase and aqueous phase at a flow rate ratio of 1:3 to 1:5 to form blank liposomes. A first hydration and a second hydration with the addition of ammonium sulfate aqueous solution are performed sequentially to achieve a final oil-water ratio of 1:6 to 1:10. After ultrafiltration to remove impurities from the external aqueous phase, doxorubicin hydrochloride solution is added to complete drug loading. This method, by optimizing microfluidic mixing parameters and a two-step hydration process, successfully prepares liposomes with uniform particle size distribution, stable internal ammonium sulfate concentration, and excellent in vitro release. Furthermore, the ultrafiltration process is bubble-free, significantly improving formulation stability, controllable drug release, and batch consistency, providing a reliable solution for the industrial production of high-quality doxorubicin hydrochloride liposomes.

Description

Technical Field

[0001] This invention belongs to the field of liposome formulations and their preparation technology, specifically relating to a doxorubicin hydrochloride liposome and its preparation method. Background Technology

[0002] Liposomes are closed vesicles formed spontaneously by lipid molecules in water, with a basic structure resembling a "sandwich" of an inner aqueous phase encapsulated by a lipid bilayer. Due to their flexibly tunable structure, liposomes can encapsulate both hydrophilic and hydrophobic drugs within the lipid bilayer, making them highly efficient and multifunctional drug delivery carriers that have attracted considerable attention. Furthermore, by optimizing lipid composition or modifying the surface, liposomes can theoretically achieve various functions such as long-term circulation, targeted delivery, reduced toxicity, and improved drug stability, making them an important research direction in the field of nanomedicine.

[0003] In terms of preparation methods, traditional processes for liposomes mainly include thin-film dispersion, reverse evaporation, ethanol injection, high-pressure homogenization, and ultrasonication. While these methods are simple and easy to implement in the laboratory, they often face problems such as low encapsulation efficiency, residual organic solvents, and poor batch-to-batch reproducibility during industrialization. To overcome these limitations, several novel preparation technologies have been developed in recent years, such as thin-film dispersion-dynamic high-pressure microfluidics, dynamic high-pressure microfluidics-freeze-thaw processes, dynamic high-pressure microfluidics-ethanol injection, heating methods, and freeze-drying methods. These methods have shown significant progress in improving encapsulation efficiency, optimizing particle size distribution, reducing solvent residues, and adapting to large-scale production, and some have already been applied in the food, cosmetics, and pharmaceutical industries.

[0004] Among various methods for preparing liposomes, microfluidics technology enables direct nano-self-assembly within chip channels by controlling the flow rate, mixing ratio, and fluid dynamics patterns of the two phases. Fluids manipulated by microfluidics possess unique properties, such as laminar flow and droplet formation. Leveraging these unique fluid behaviors, a series of microparticle fabrications that are difficult to achieve using conventional methods can be accomplished, including enhanced fluid mixing and intervention in nanostructure assembly at the microscopic level, demonstrating unique advantages.

[0005] Doxorubicin, also known as doxorubicin, is an anthracycline broad-spectrum antitumor antibiotic and a cytotoxic antimitotic agent capable of inducing remission in various malignant tumors. However, its clinical application is limited due to severe cardiotoxicity and dose-limiting side effects. To overcome this problem, the first nanoliposome formulation, doxorubicin hydrochloride liposome injection (trade name: Doxil / Caelyx), was approved by the US FDA in 1995. Its current indications include the treatment of ovarian cancer, HIV-related Kaposi's sarcoma, and multiple myeloma. This formulation significantly improves the drug's pharmacokinetic properties through liposome encapsulation and achieves targeted accumulation in tumor tissues through enhanced permeation and retention (EPR effect).

[0006] According to the European Medicines Agency's review report and practical application requirements for the original drug Caelyx, doxorubicin hydrochloride liposomes, doxorubicin hydrochloride liposome injection is a sterile injection solution, requiring sterilization through filtration to ensure product sterility. The main preparation process involves first mixing the water and oil phases at high temperature, then extruding to obtain blank liposomes, followed by ultrafiltration to form an ammonium sulfate gradient, and finally loading the active drug. Its core product characteristics include an average particle size of 80nm-100nm, a release rate of not less than 60% after 3 hours, and PDI-related data that have not yet been disclosed.

[0007] Liposome particle size is a crucial factor influencing their aggregation behavior at tumor sites. The Polydispersity Index (PDI) is a core indicator measuring the width and degree of particle size distribution, widely used in nanomaterials, colloid science, and other fields. A PDI value closer to 0 indicates a narrower particle size distribution, more uniform size, and better dispersibility, making it easier to filter and sterilize. Conversely, a PDI value closer to 1 indicates a wider particle size distribution, greater particle variation, and poorer dispersibility, while also posing greater challenges to filtration and sterilization. For doxorubicin hydrochloride liposomes, a uniform particle size not only improves the product's physical and chemical stability, ensuring predictable and controllable drug release at the target site, but also guarantees targeted delivery efficiency and bioavailability. This is because particle size directly affects the interaction between liposomes and the body, as well as the ability of tumor cells to reach the target site. Furthermore, batch-to-batch PDI consistency is key to achieving repeatable treatment effects and clinical efficacy. Therefore, effectively controlling the PDI < 0.1 (generally considered the standard for high monodispersity) will help ensure smooth sterilization of doxorubicin hydrochloride liposomes, improve formulation stability, achieve controlled drug release and targeted delivery, and maintain batch quality consistency.

[0008] The concentration of ammonium sulfate in the aqueous phase is a key indicator for assessing the stability of doxorubicin hydrochloride liposomes and a core control point for formulation. This concentration affects the encapsulation state of the drug, directly influencing drug release and leakage, and ultimately impacting the clinical efficacy and safety of the product. Specifically, the state of doxorubicin hydrochloride within the lipid bilayer depends on the ammonium sulfate encapsulation concentration. Excessively high concentrations of ammonium sulfate lead to firm precipitation of doxorubicin hydrochloride within the lipid bilayer, resulting in slow or even no drug release. Conversely, excessively low concentrations prevent stable precipitation of doxorubicin hydrochloride within the lipid bilayer, leading to low encapsulation efficiency or drug leakage. Therefore, controlling the concentration of ammonium sulfate in the aqueous phase within a reasonable range helps ensure stable drug release in vivo and avoids the safety risks associated with premature drug leakage. According to literature reports and practical experience, the effective concentration of ammonium sulfate in the aqueous phase required for liposome stability is approximately or slightly higher than 0.6 mg / ml.

[0009] Release rate is an evaluation method for the in vivo release of liposomal drugs when simulated in vitro in vitro, similar to clinical drug use. Its purpose is to ensure consistent drug release characteristics across different batches of products in vivo, and it is also a crucial indicator for liposomal product quality control. This indicator is affected not only by particle size and particle size distribution but also by ammonium sulfate concentration. The original formulation, Caelyx, launched in China in 2005, uses an ammonium chloride-histidine medium to detect the in vitro release of doxorubicin hydrochloride liposomes, with an acceptable standard of at least 60% for in vitro release over 3 hours.

[0010] Existing patents mostly employ an ethanol injection method combined with an ammonium sulfate gradient method to prepare doxorubicin hydrochloride liposomes. Patents CN100376249C and CN100431525C use similar preparation methods, both involving dissolving lipid components in an alcohol solvent to form a mixture, mixing this mixture with an aqueous ammonium sulfate solution, and then subjecting the resulting mixture to pore extrusion to obtain a liposome suspension. This process requires two stages of pore extrusion and 10 filtrations, making it complex. Other patents use a continuous flow process, such as CN119280169A, but this process has significant shortcomings. The particle size and particle size distribution (PDI) of the prepared liposomes differ significantly from the original product, failing to meet the in vitro consistency PBE equivalence requirements. Many examples have a PDI higher than 0.2, while only a few examples have a PDI lower than 0.1 but with low encapsulation efficiency, making it difficult to consistently prepare high-quality liposomes. Furthermore, significant challenges arise in the filtration and sterilization process. This patent's examples do not disclose the concentration of ammonium sulfate in the aqueous phase of the doxorubicin hydrochloride liposomes, nor do they disclose in vitro release data. Patent CN111214438A uses microfluidic technology to prepare doxorubicin micelles. The particle size distribution (PDI) of the prepared product is relatively large. No implementation cases with PDI below 0.1 have been found, and no product release evaluation data have been disclosed. Summary of the Invention

[0011] To address the shortcomings of existing technologies, this invention provides doxorubicin hydrochloride liposomes and their preparation method, aiming to improve the overall performance and process stability of the formulation.

[0012] This invention provides a method for preparing doxorubicin hydrochloride liposomes, comprising the following steps:

[0013] (1) Prepare an aqueous solution of ammonium sulfate and an oil phase solution, wherein the oil phase solution contains lipid components and an organic solvent; the concentration of the aqueous solution of ammonium sulfate is 30-36 mg / mL; the lipid components in the oil phase solution include hydrogenated soybean phosphatidylcholine, cholesterol and polyethylene glycol-distearate phosphatidylethanolamine, and the total concentration of lipid components in the oil phase solution is 25-35 mg / mL;

[0014] (2) The oil phase solution and the ammonium sulfate aqueous solution are mixed using microfluidic technology to form a blank liposome mixture, wherein the flow rate ratio of the oil phase to the water phase is 1:3 to 1:5;

[0015] (3) The mixture obtained in step (2) is subjected to a first hydration at a temperature of 60-70℃ for 0.5-2 hours.

[0016] (4) Add ammonium sulfate aqueous solution to the mixture after hydration in step (3) for a second hydration. The hydration temperature is 60-70℃ and the hydration time is 0.5-2 hours. The oil-water ratio in the system after the addition is 1:6 to 1:10.

[0017] (5) The mixture obtained in step (4) is subjected to ultrafiltration to remove ammonium sulfate and organic solvents in the aqueous phase;

[0018] (6) Add doxorubicin hydrochloride solution to the mixture after ultrafiltration in step (5) and load the drug at 55-65℃ to obtain doxorubicin hydrochloride liposomes.

[0019] As a further optimization of the method for preparing doxorubicin hydrochloride liposomes, in step (1), the organic solvent is ethanol.

[0020] As a further optimization of the method for preparing doxorubicin hydrochloride liposomes, in step (2), the oil phase solution and the ammonium sulfate aqueous solution are mixed by a microreactor using microfluidic technology, wherein the oil phase solution and the ammonium sulfate aqueous solution are mixed through a common channel flowing through the microreactor.

[0021] As a further optimization of the preparation method of doxorubicin hydrochloride liposomes, the hydration temperature in steps (3) and (4) is 65±2℃.

[0022] As a further optimization of the preparation method of doxorubicin hydrochloride liposomes, the temperature of the ammonium sulfate aqueous solution added in step (4) is 60-70℃.

[0023] As a further optimization of the method for preparing doxorubicin hydrochloride liposomes, the concentration of the doxorubicin hydrochloride solution in step (6) is 8-12 mg / ml, and the amount added is such that the mass ratio of doxorubicin hydrochloride to lipid components is 1:3 to 1:5, preferably 1:3.5 to 1:4.5.

[0024] As a further optimization of the preparation method of doxorubicin hydrochloride liposomes, the drug loading temperature in step (6) is 60±2℃.

[0025] As a further optimization of the method for preparing doxorubicin hydrochloride liposomes, in step (1), the mass ratio of hydrogenated soybean phosphatidylcholine, cholesterol and polyethylene glycol-distearate phosphatidylethanolamine is (2.5-3.5):(0.9-1.1):1.

[0026] As a further optimization of the method for preparing doxorubicin hydrochloride liposomes, in step (1), the organic solvent is ethanol, and the mass ratio of hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearate phosphatidylethanolamine to ethanol is (17-21):(5.8-7.0):(5.8-7.0):100.

[0027] Based on the method for preparing doxorubicin hydrochloride liposomes, the present invention further provides a doxorubicin hydrochloride liposome, which is prepared by any of the methods described above.

[0028] Beneficial effects

[0029] Compared with existing technologies, this invention provides a novel method for preparing doxorubicin hydrochloride liposomes, successfully producing doxorubicin hydrochloride liposomes with uniform particle size distribution (PDI < 0.1), stable ammonium sulfate concentration in the aqueous phase (0.66–0.73 mg / ml), satisfactory in vitro release (≥75% release rate at 3 hours), and smooth, bubble-free ultrafiltration. This method effectively solves common problems in traditional methods, such as poor particle size distribution, unstable drug release, fluctuations in aqueous phase encapsulation efficiency, and bubble generation during production. It helps improve the physicochemical stability of the formulation, controllable drug release, and targeted delivery efficiency, and also helps ensure batch-to-batch quality consistency, providing a reliable and reproducible technical path for the industrial production of high-quality doxorubicin hydrochloride liposomes. Detailed Implementation

[0030] The present invention is further illustrated below with specific embodiments. These embodiments are exemplary and intended to illustrate the problem and explain the present invention, and are not intended to be limiting.

[0031] Comparative Example 1

[0032] Ammonium sulfate was weighed and added to a measured amount of purified water. After stirring and dissolving, a 33 mg / ml ammonium sulfate solution was obtained and incubated in a water bath at 65±2℃ for later use. HSPC, CHO-HP, and DSPE-mPEG2000 were weighed separately and added to a measured amount of anhydrous ethanol. The solution was incubated in a water bath at 65±2℃ until dissolved, yielding an oil phase solution for later use. Blank liposomes were prepared by mixing the oil and aqueous phases in a microreactor at flow rates of 17 ml / min and 136 ml / min (oil-to-water ratio 1:8) using an infusion pump. After mixing, the mixture was hydrated at 65℃ for 1 h, cooled to room temperature, and ammonium sulfate and solution in the outer aqueous phase were removed by tangential flow ultrafiltration. A 10 mg / ml doxorubicin hydrochloride solution was added at a ratio of 4:1, and the mixture was loaded with the drug at 60℃ to obtain a 2 mg / ml doxorubicin hydrochloride liposome injection solution. The solution was then purged with nitrogen and stored at 2-8℃. Among them, HSPC is hydrogenated soybean phosphatidylcholine; CHO-HP is high-purity injection-grade cholesterol; and DSPE-mPEG2000 is polyethylene glycol 2000-distearate phosphatidylethanolamine.

[0033] Comparative Example 2

[0034] Weigh ammonium sulfate and add it to a measured amount of purified water. Stir to dissolve and obtain a 33 mg / ml ammonium sulfate solution. Incubate the solution in a water bath at 65±2℃ for later use. Weigh HSPC, CHO-HP, and DSPE-mPEG2000 separately and add them to a measured amount of anhydrous ethanol (HSPC:CHO-HP:DSPE-mPEG2000:ethanol = 95.8:31.9:31.9:500). Incubate the solution in a water bath at 65±2℃ until dissolved to obtain an oil phase solution for later use. Blank liposomes were prepared by mixing the oil and aqueous phases in a microreactor using an infusion pump at flow rates of 22 ml / min and 132 ml / min (oil-to-water ratio 1:6). After mixing, the mixture was hydrated at 65°C for 1 h, cooled to room temperature, and ammonium sulfate and solution in the outer aqueous phase were removed by tangential flow ultrafiltration. Doxorubicin hydrochloride solution of 10 mg / ml was added at a ratio of 4:1, and the mixture was loaded with the drug at 60°C to obtain a 2 mg / ml doxorubicin hydrochloride liposome injection solution. The solution was then purged with nitrogen and stored at 2-8°C.

[0035] Comparative Example 3

[0036] Weigh ammonium sulfate and add it to a measured amount of purified water. Stir to dissolve and obtain a 33 mg / ml ammonium sulfate solution. Incubate the solution in a water bath at 65±2℃ for later use. Weigh HSPC, CHO-HP, and DSPE-mPEG2000 separately and add them to a measured amount of anhydrous ethanol (HSPC:CHO-HP:DSPE-mPEG2000:ethanol = 95.8:31.9:31.9:500). Incubate the solution in a water bath at 65±2℃ until dissolved to obtain an oil phase solution for later use. The oil and aqueous phases were mixed in a microreactor using an infusion pump at flow rates of 38 ml / min and 152 ml / min (oil-to-water ratio 1:4) to prepare blank liposomes. After mixing, the mixture was hydrated at 65°C for 1 h, cooled to room temperature, and ammonium sulfate and solution in the aqueous phase were removed by tangential flow ultrafiltration. A large number of bubbles were generated during the ultrafiltration process. After the bubbles disappeared, 10 mg / ml doxorubicin hydrochloride solution was added at a ratio of 4:1. The mixture was loaded with the drug at 60°C to obtain 2 mg / ml doxorubicin hydrochloride liposome injection solution. The solution was then purged with nitrogen and stored at 2-8°C.

[0037] Comparative Example 4

[0038] Weigh ammonium sulfate and add it to a measured amount of purified water. Stir to dissolve and obtain a 33 mg / ml ammonium sulfate solution. Incubate the solution in a water bath at 65±2℃ for later use. Weigh HSPC, CHO-HP, and DSPE-mPEG2000 separately and add them to a measured amount of anhydrous ethanol (HSPC:CHO-HP:DSPE-mPEG2000:ethanol = 95.8:31.9:31.9:500). Incubate the solution in a water bath at 65±2℃ until dissolved to obtain an oil phase solution for later use. The oil and aqueous phases were mixed in a microreactor using an infusion pump at flow rates of 66 ml / min and 132 ml / min (oil-to-water ratio 1:2) to prepare blank liposomes. Numerous bubbles were generated, potentially forming multilayer liposomes (MLVs). The mixture was then hydrated in a water bath at 65°C for 1 hour. 23.06 g of the blank liposomes was removed, and 49.41 g of hot ammonium sulfate solution was added (oil-to-water ratio approximately 1:8). After gentle shaking, the mixture was hydrated in a water bath at 65°C for 1 hour, then cooled to room temperature. Ammonium sulfate and ethanol in the aqueous phase were removed by tangential flow ultrafiltration. A 10 mg / ml doxorubicin hydrochloride solution was added at a 4:1 ratio, and the mixture was loaded at 60°C to obtain a 2 mg / ml doxorubicin hydrochloride liposome injection solution. The solution was then nitrogen-purged and stored at 2-8°C.

[0039] Example 1

[0040] Weigh ammonium sulfate and add it to weighed purified water. After stirring and dissolving, a 33 mg / ml ammonium sulfate solution is obtained and incubated in a water bath at 65±2℃ for later use. Weigh HSPC, CHO-HP, and DSPE-mPEG2000 separately and add them to weighed anhydrous ethanol (HSPC:CHO-HP:DSPE-mPEG2000:ethanol = 95.8:31.9:31.9:500). Incubate in a water bath at 65±2℃ until dissolved to obtain an oil phase solution for later use. Set up an infusion pump to mix the oil phase and aqueous phase in a microreactor at flow rates of 38 ml / min and 152 ml / min (oil-water ratio 1:4) to prepare blank liposomes. Incubate in a water bath at 65℃ for 1 h. Take out 59.3 g of blank liposomes and add 24.7 g of hot ammonium sulfate solution (oil-water ratio approximately 1:6 at this point). Shake gently and incubate in a water bath at 65℃ for 1 h. Cool to room temperature and record as sample A. Another 59.3g of blank lipid was added to 49.4g of ammonium sulfate solution (the oil-to-water ratio was approximately 1:8). After gentle shaking, the mixture was placed in a water bath at 65°C for 1 hour to hydrate again. The solution was then cooled to room temperature and designated as sample B. Samples A and B were subjected to tangential flow ultrafiltration to remove the ammonium sulfate and solution from the external aqueous phase. 10mg / ml doxorubicin hydrochloride solution was added to both samples at a ratio of 4:1. The mixture was loaded with the drug at 60°C to prepare a 2mg / ml doxorubicin hydrochloride liposome injection solution. The solution was then nitrogen-purged and stored at 2-8°C.

[0041] Example 2

[0042] Weigh ammonium sulfate and add it to weighed purified water. After stirring and dissolving, a 33 mg / ml ammonium sulfate solution is obtained and incubated in a water bath at 65±2℃ for later use. Weigh HSPC, CHO-HP, and DSPE-mPEG2000 separately and add them to weighed anhydrous ethanol (HSPC:CHO-HP:DSPE-mPEG2000:ethanol = 95.8:31.9:31.9:500). Incubate in a water bath at 65±2℃ until dissolved to obtain an oil phase solution for later use. Set up an infusion pump to mix the oil phase and aqueous phase in a microreactor at flow rates of 50 ml / min and 150 ml / min (oil-water ratio 1:3) to prepare blank liposomes. Incubate in a water bath at 65℃ for 1 h. Take out 46.95 g of blank liposomes and add 86.46 g of hot ammonium sulfate solution (oil-water ratio approximately 1:10 at this point). Shake gently and incubate in a water bath at 65℃ for 1 h. Cool to room temperature and record as sample A. Another 46.95g of blank lipid was added to 37.06g of ammonium sulfate solution (the oil-water ratio was approximately 1:6). After gentle shaking, the sample was placed in a water bath at 65°C for 1 hour to hydrate again. The sample was then cooled to room temperature and designated as sample B. Samples A and B were subjected to tangential flow ultrafiltration to remove the ammonium sulfate and solution from the external aqueous phase. 10mg / ml doxorubicin hydrochloride solution was added at a ratio of 4:1. The sample was loaded with the drug at 60°C to obtain a 2mg / ml doxorubicin hydrochloride liposome injection solution. The solution was then nitrogen-filled and stored at 2-8°C.

[0043] Example 3

[0044] Weigh ammonium sulfate and add it to weighed purified water. After stirring and dissolving, a 33 mg / ml ammonium sulfate solution is obtained and incubated in a water bath at 65±2℃ for later use. Weigh HSPC, CHO-HP, and DSPE-mPEG2000 separately and add them to weighed anhydrous ethanol (HSPC:CHO-HP:DSPE-mPEG2000:ethanol = 95.8:31.9:31.9:500). Incubate in a water bath at 65±2℃ until dissolved to obtain an oil phase solution for later use. Set up an infusion pump to mix the oil phase and aqueous phase in a microreactor at flow rates of 32 ml / min and 160 ml / min (oil-water ratio 1:5) to prepare blank liposomes. Incubate in a water bath at 65℃ for 1 h. Take out 48.95 g of blank liposomes and add 8.42 g of hot ammonium sulfate solution (oil-water ratio approximately 1:6 at this point). Shake gently and incubate in a water bath at 65℃ for 1 h, then cool to room temperature. Ammonium sulfate and solution in the external aqueous phase were removed by tangential flow ultrafiltration. Doxorubicin hydrochloride solution of 10 mg / ml was added at a ratio of 4:1. The drug was loaded at 60°C to prepare 2 mg / ml doxorubicin hydrochloride liposome injection. Nitrogen was added and the solution was stored at 2-8°C.

[0045] Test case

[0046] For all the doxorubicin hydrochloride liposome samples prepared in the examples and comparative examples, the particle size distribution (PDI) was determined by a nanoparticle size analyzer, the ammonium sulfate content in the finished product was detected by ion chromatography, and the in vitro release rate at 3 h was determined by liquid chromatography. At the same time, the smoothness of the ultrafiltration process and the operation status such as whether bubbles were generated were recorded in real time. The above test results and operation status are summarized in Table 1.

[0047] Table 1

[0048]

[0049] As shown in Table 1, Comparative Examples 1, 2, and 3 prepared doxorubicin hydrochloride liposomes using a traditional single-stage hydration process. Although the key microfluidic parameter of the oil-water ratio (FRR) was adjusted extensively (1:8, 1:6, and 1:4, respectively), the FRR directly affects the fluid dynamics within the microchannels, thus significantly influencing the formation and shape of the initial liposomes. However, none of these methods simultaneously met the quality control requirements of PDI < 0.1, ammonium sulfate content 0.6–0.8 mg / ml, 3-hour release ≥ 60%, and no bubbles during ultrafiltration. Comparative Examples 1 and 2 both had PDIs higher than 0.1, failing to meet the monodispersity requirement. While Comparative Example 3 achieved a lower PDI (0.030), its release was significantly lower (49%), and a large number of bubbles appeared during ultrafiltration, indicating inherent defects in its process stability and product performance. These results demonstrate that under single hydration conditions, simply adjusting the oil-water ratio is insufficient to comprehensively optimize the physicochemical properties and process behavior of liposomes to meet the quality control requirements.

[0050] In contrast, Examples 1, 2, and 3 (comprising a total of 5 samples) employed a two-stage hydration process. After initial microfluidic mixing and the first hydration, a second hydration was performed by adding a certain amount of hot ammonium sulfate solution before further processing. In these examples, when the initial oil-to-water ratio was in the range of 1:3 to 1:5, and the second hydration adjusted the oil-to-water ratio to 1:6 to 1:10, all samples achieved an optimal average particle size slightly larger than 80 nm, a PDI below 0.1, an ammonium sulfate content between 0.66 and 0.73 mg / ml, a 3-hour release rate exceeding 75%, and a smooth, bubble-free ultrafiltration process. These results demonstrate that, under the synergistic control of specific parameters, a two-stage hydration process can synergistically improve the particle size uniformity of liposomes, the encapsulation efficiency of the internal aqueous phase, and drug release characteristics, thereby comprehensively meeting product quality standards. Comparative Example 4 also employed a two-stage hydration process, but the initial oil-to-water ratio was set at 1:2. Although it was eventually adjusted to 1:8, this still resulted in a low ammonium sulfate content (0.48 mg / ml) and insufficient release (42%). This indicates that, in addition to the two-stage hydration process, specific process control steps also play a crucial role in sample preparation.

[0051] Further research revealed that in Examples 1, 2, and 3, the addition of the external aqueous phase during the second hydration phase resulted in a final oil-to-water ratio covering a wide range of 1:6 to 1:10. However, the ammonium sulfate content in the internal aqueous phase of all samples remained stable within the range of 0.66 to 0.73 mg / ml, with minimal batch-to-batch fluctuations. Based on this experimental result, it can be concluded that the preparation method used in this invention does not significantly alter the ammonium sulfate concentration level in the aqueous phase within the liposomes during the second hydration phase with the addition of the external aqueous phase ammonium sulfate solution. One possible explanation for the underlying mechanism of this finding is that ammonium sulfate ionizes in aqueous solution to form highly polar, highly water-soluble hydrated ammonium ions and hydrated sulfate ions. The middle region of the phospholipid bilayer of the liposome is a hydrophobic environment, lacking affinity for water-soluble ions. This hydrophobic region effectively hinders ion permeability, making it difficult for ions in the external aqueous phase to cross the hydrophobic barrier and enter the liposome interior. Therefore, the process of the present invention can flexibly adjust the oil-water ratio by adding an external aqueous phase to optimize the particle size distribution, while avoiding fluctuations in the concentration of ammonium sulfate in the internal aqueous phase. This also forms the basis for the stable encapsulation and controllable release of doxorubicin hydrochloride.

[0052] It is worth noting that the above experimental results also reveal that bubble formation is a significant hidden factor restricting the quality of doxorubicin hydrochloride liposomes. In microfluidic technology, the flow rate ratio (FRR) of the oil and aqueous phases usually needs to be as close as possible to ensure system stability. The phospholipid components HSPC and MPEG-2000-DSPE used in this invention both have amphiphilic characteristics and can act as surfactants in the system. When the oil phase ratio is too high (e.g., FRR = 1:4 in Comparative Example 3, initial FRR = 1:2 in Comparative Example 4), the excess oil phase component and the surfactant-like effect of the phospholipids superimpose, disrupting the spontaneous assembly equilibrium of the lipid bilayer. This leads to decreased stability of the liposomes during the microfluidic mixing stage or subsequent ultrafiltration, resulting in the generation of a large number of bubbles. The formation of these bubbles further induces the generation of MLVs, which not only directly leads to a wider particle size distribution (as in Comparative Example 4, although the FRR was adjusted by secondary hydration, the MLVs formed in the initial stage were difficult to completely eliminate, and the final release rate was still only 42%), but also hinders the smooth progress of the filtration and sterilization process. Simultaneously, it disrupts the stable encapsulation state of the drug within the liposomes, causing problems such as substandard release rates. Although Comparative Example 3 achieved a lower PDI by adjusting the FRR, the encapsulation environment disturbance caused by the bubbles still resulted in the release rate not meeting the standard.

[0053] In summary, the preparation method of the present invention, by combining a two-stage hydration process with an optimized parameter group, effectively solves common problems in the microfluidic preparation of doxorubicin hydrochloride liposomes, such as uneven particle size distribution, unsatisfactory encapsulation of the internal aqueous phase, low release rate, and numerous process bubbles. This provides a feasible path for the industrial and stable production of high-quality doxorubicin hydrochloride liposome formulations.

[0054] The above embodiments are exemplary and are intended to illustrate the technical concept and features of the present invention, so that those skilled in the art can understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing liposomal doxorubicin hydrochloride, characterized by, The method comprises the following steps: (1) preparing an aqueous ammonium sulfate solution and an oil phase solution, wherein the concentration of the aqueous ammonium sulfate solution is 30-36 mg / ml; the oil phase solution comprises lipid components and an organic solvent; the lipid components in the oil phase solution comprise hydrogenated soybean phosphatidylcholine, cholesterol and polyethylene glycol-distearyl phosphatidyl ethanolamine, and the total concentration of the lipid components in the oil phase solution is 25-35 mg / ml; (2) mixing the oil phase solution and the aqueous ammonium sulfate solution by using a microfluidic technology to form a blank liposome mixture, wherein the flow rate ratio of the oil phase to the aqueous phase is 1:3 to 1:5; (3) performing first hydration on the mixture obtained in step (2) at a temperature of 60-70℃ for 0.5-2 hours; (4) adding the aqueous ammonium sulfate solution to the mixture after the hydration in step (3) to perform second hydration at a temperature of 60-70℃ for 0.5-2 hours, wherein the oil-water ratio in the system after the addition is 1:6 to 1:10; (5) performing ultrafiltration on the mixture obtained in step (4) to remove the external aqueous phase ammonium sulfate and the organic solvent; (6) adding a doxorubicin hydrochloride solution to the mixture after the ultrafiltration in step (5) to perform drug loading at 55-65℃ to obtain doxorubicin hydrochloride liposomes; In step (2), the mixing of the oil phase solution and the aqueous ammonium sulfate solution by using the microfluidic technology is realized by a microreactor, and the oil phase solution and the aqueous ammonium sulfate solution are mixed by flowing through the channel of the microreactor together; In step (1), the organic solvent is ethanol, and the mass ratio of hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearyl phosphatidyl ethanolamine to ethanol is (17-21):(5.8-7.0):(5.8-7.0):

100.

2. The production method according to claim 1, characterized by, The hydration temperature in steps (3) and (4) is 65±2℃.

3. The method of claim 1, wherein, The temperature of the added aqueous ammonium sulfate solution in step (4) is 60-70℃.

4. The method of claim 1, wherein, In step (6), the concentration of the doxorubicin hydrochloride solution is 8-12 mg / ml, and the amount of addition is such that the mass ratio of doxorubicin hydrochloride to lipid components is 1:3 to 1:

5.

5. The preparation method according to claim 1, characterized in that, The drug loading temperature in step (6) is 60±2℃.

6. A liposomal doxorubicin hydrochloride characterized in that, The doxorubicin hydrochloride liposomes are prepared by the method of any one of claims 1-5.

Images