Baicalein liposome injection and preparation method and application thereof

Abstract

This invention provides a baicalein liposome injection, its preparation method, and its application. The baicalein liposome comprises phospholipid bilayer liposome vesicles and baicalein embedded in the hydrophobic phospholipid layer of the liposomes. The phospholipid bilayer liposome vesicle membrane material comprises egg yolk lecithin and cholesterol. The egg yolk lecithin contains ≥98% phosphatidylcholine (PC) by mass. The average hydrated particle size of the baicalein liposomes is 50~150 nm, the PDI is 0.10~0.30, the baicalein encapsulation efficiency is ≥85%, and the baicalein drug loading is ≥10%. By optimizing the membrane hydration preparation process, the encapsulation efficiency, drug loading, and colloidal storage stability of the baicalein liposomes are effectively improved, which can significantly improve the in vivo bioavailability and tumor-targeting enrichment ability of baicalein, achieving a highly efficient and broad-spectrum inhibitory effect on various solid tumors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, specifically to baicalin liposome injection, its preparation method, and its application. Background Technology

[0002] Baicalein (molecular formula C) 15 H 10 O5, CAS No. 491-67-8, chemical name 5,6,7-trihydroxyflavone (also commonly known as baicalin), is mainly extracted from the root of *Scutellaria baicalensis*, a plant belonging to the genus *Scutellaria* in the Lamiaceae family. Extensive pharmacological studies have confirmed that baicalin is a highly active natural flavonoid compound with multiple pharmacological effects, including antioxidant, anti-allergic, antiviral, and antitumor properties. It exerts its anticancer effects through multiple pathways, such as inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and enhancing the body's immunity. Due to its safe and low-toxicity natural drug advantages, it has become a popular raw material for the development of targeted antitumor agents. Meanwhile, liposome delivery systems, as a mature drug carrier technology, have been widely used in the preparation of clinical chemotherapy drugs, and several antitumor liposome drugs have achieved commercial application, with their clinical efficacy and market application value fully validated.

[0003] In actual drug development and clinical application, the inherent characteristics of natural baicalin and traditional administration methods present several unavoidable drawbacks. First, baicalin is a poorly water-soluble flavonoid with extremely low solubility in water, and its content in natural Scutellaria baicalensis root is very low, only 0.04%~0.28%. Industrially, it requires acid or enzymatic hydrolysis to break down baicalin, resulting in high raw material costs. Second, under traditional oral administration, baicalin has low oral bioavailability, rapid metabolism after entering the body, and short retention time, making it difficult to sustain its anti-tumor efficacy. These inherent defects directly prevent the full realization of baicalin's pharmacological activity, greatly limiting its practical application in anti-tumor clinical treatment.

[0004] To address the issues of poor water solubility, rapid in vivo metabolism, and insufficient efficacy of baicalein, the mainstream solution in the industry is to encapsulate and modify baicalein using liposome delivery systems. Liposomes are artificially prepared bilayer phospholipid vesicle carriers that can fundamentally optimize the drug delivery performance of baicalein, specifically achieving three major technological improvements: first, significantly enhancing the water solubility and in vitro stability of baicalein, prolonging its retention time in the human bloodstream; second, achieving passive targeted enrichment through the osmotic retention effect of tumor tissue, effectively increasing the drug concentration at the tumor lesion site; and third, smoothly releasing the drug through sustained-release and controlled-release mechanisms, reducing the drug's toxic side effects on normal organisms. Currently, various baicalein liposome research and development trials are underway in the industry. Researchers are optimizing the formulation structure by using different phospholipid raw materials, polymer modification materials, and preparation processes. Simultaneously, PEG (Polyethylene Glycol) modification and polysaccharide modification technologies are also widely used to optimize the targeting and stability of baicalein liposomes, thereby improving its anti-tumor application efficacy.

[0005] Although existing liposome technology can initially improve the delivery defects of baicalein, current liposome formulations of baicalein prepared through various studies still have significant technical shortcomings and have not yet met the standards for industrialization and clinical application. According to the Chinese Pharmacopoeia, qualified liposome formulations must meet the requirements of an encapsulation efficiency of not less than 80% and a drug loading of not less than 10%. However, the parameters of existing research products are far below these standards. For example, some formulations have a drug loading of only 3.36% or 6.1%, or an encapsulation efficiency of only 32%, indicating extremely poor drug loading performance. At the same time, the existing efficacy evaluation system has obvious limitations, mostly conducting in vitro experiments on single tumor cells, lacking systematic efficacy and safety verification at the multi-cancer cell and in vivo animal levels. In addition, existing related formulations are mostly for metabolic regulation and antibacterial purposes, and are only suitable for intraperitoneal injection, not standardized antitumor injectable dosage forms. Furthermore, key parameters such as particle size, drug loading, and encapsulation efficiency cannot be precisely controlled, resulting in insufficient overall formulation stability and applicability, making it difficult to meet the needs of standardized clinical antitumor treatment for multiple cancer types. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a baicalin liposome injection, its preparation method, and its applications. By optimizing the thin-film hydration preparation process, limiting the use of egg yolk lecithin membrane material with high phosphatidylcholine purity, and controlling the feeding ratio of baicalin, egg yolk lecithin, and cholesterol, combined with an integrated improved process of high-vacuum delayed rotary evaporation for solvent removal, high-temperature hydration, and pulsed ultrasonic homogenization, the encapsulation efficiency, drug loading, and colloidal storage stability of baicalin liposomes are effectively improved. This significantly enhances the in vivo bioavailability and tumor-targeting accumulation ability of baicalin, achieving highly efficient and broad-spectrum inhibitory effects against various solid tumors.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a baicalin liposome, comprising phospholipid bilayer liposome vesicles and baicalin embedded in the hydrophobic phospholipid layer of the liposome; the phospholipid bilayer liposome vesicle membrane material comprises egg yolk lecithin and cholesterol; the egg yolk lecithin contains ≥98% phosphatidylcholine (PC) by mass; the baicalin liposome has an average hydrated particle size of 50~150 nm, a PDI of 0.10~0.30, a baicalin encapsulation efficiency of ≥85%, and a baicalin drug loading of ≥10%.

[0008] The present invention also provides a basidiosomal liposome lyophilized formulation, wherein the basidiosomal liposome lyophilized formulation is prepared by vacuum freeze-drying of the above-mentioned basidiosomal liposomes and a lyophilization excipient.

[0009] Furthermore, the lyophilization excipient is glucose with a concentration of 3% to 5%.

[0010] The present invention also provides a baicalein liposome injection, wherein the baicalein liposome injection is prepared by the above-mentioned baicalein liposome or the above-mentioned lyophilized baicalein liposome preparation and an aqueous injection medium, and the final concentration of baicalein in the preparation is 4.5~7.5 mg / mL.

[0011] This invention also provides a method for preparing baicalein liposome injection, comprising the following steps: Baicalein, egg yolk lecithin, and cholesterol are mixed at a weight ratio of 1:3:0.8~1.5, and then dissolved completely in an organic solvent to obtain a mixture solution. The mixture solution was subjected to vacuum rotary evaporation, with the rotary evaporation vacuum degree controlled at ≥0.08MPa and the water bath temperature at 37~40℃. After the lipid film was formed, the rotary evaporation was continued for 15~35min under the same conditions to obtain a dense lipid film. When preparing liquid injection dosage forms, an aqueous medium for injection is added to the lipid film, followed by hydration and homogenization treatment to obtain a uniformly dispersed crude dispersion of liposomes. When preparing lyophilized injection formulations, a glucose aqueous solution with a mass concentration of 3% to 5% is added to the lipid film, followed by hydration and homogenization treatment to obtain a uniformly dispersed crude dispersion of liposomes. The obtained crude liposome dispersion was filtered through a sterile microporous membrane for sterilization. After filtration in liquid form, baicalein liposome injection was obtained. After filtration in lyophilized form, it was subjected to vacuum freeze-drying to obtain lyophilized baicalein liposome preparation. The lyophilized baicalein liposome preparation was mixed with an aqueous medium for injection and shaken to obtain lyophilized baicalein liposome injection.

[0012] Furthermore, the hydration and homogenization process specifically involves: firstly, using a conventional ultrasonic cleaner to hydrate the lipid film in a water bath at 37~40℃, and then using a non-contact ultrasonic cell disruptor for pulse homogenization, with the ultrasonic working mode at 900W, ultrasonic for 5 seconds, and intermittent for 5 seconds.

[0013] Furthermore, the filtration and sterilization process uses a sterile microporous filter membrane with a pore size ≤0.3μm.

[0014] Furthermore, the storage conditions for the finished product are as follows: liquid dosage forms should be stored at 2~8℃ away from light, and lyophilized dosage forms should be stored at 4℃~-80℃ in a sealed container away from light.

[0015] The present invention also provides the application of baicalin liposome injection in the preparation of a broad-spectrum antitumor drug, wherein the antitumor drug is an intravenously administered preparation and is applicable to the treatment of tumors including breast cancer, ovarian cancer, lung cancer, and colorectal cancer.

[0016] Furthermore, the baicalin liposome injection is used to prepare an anti-ovarian cancer drug.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a baicalin liposome injection with an encapsulation efficiency of over 95% and a stable drug loading of ≥10%. The optimal sample achieved an encapsulation efficiency of 97.3% and a drug loading of 12.5%, far exceeding the current research level in the industry. Simultaneously, the average hydrated particle size of the formulation is stably controlled at around 90.5 nm, with a PDI of 0.10~0.30. The particles are uniformly dispersed and the colloidal state is stable. After standing at 4℃ for 30 days, there is no stratification, precipitation, or drug separation. This effectively solves the problems of poor solubility, weak stability, and low effective drug loading in traditional formulations, significantly improving the in vivo action of baicalin and meeting the pharmaceutical quality requirements for clinical intravenous injection.

[0018] Furthermore, this invention uses injection-grade refined egg yolk lecithin with a phosphatidylcholine (PC) content ≥98% as the membrane material. This differs from conventional 80% low-purity phospholipids, which are prone to forming flocculation and turbidity. It can form a complete phospholipid bilayer with good light transmittance and a regular structure. Simultaneously, the ratio of baicalin, egg yolk lecithin, and cholesterol is controlled at 1:3:0.8~1.5. This ensures the stability of the rigid structure of the phospholipid membrane while preventing excessive cholesterol from crowding out drug loading sites and reducing efficacy. This invention is compatible with both liquid injection and lyophilized dosage forms. The lyophilized product can be stored for a long time at 4℃ to -80℃. After reconstitution, there is no significant decrease in particle size, encapsulation efficiency, or drug loading, effectively solving the problem of short storage periods for liquid formulations and significantly expanding the storage, transportation, and clinical application scenarios of the formulation.

[0019] This invention provides a method for preparing baicalein liposome injection. Under high vacuum conditions above 0.08 MPa and a water bath at 37-40°C, after film formation, continuous rotary evaporation for 15-35 minutes removes residual trace amounts of organic solvent, resulting in a dense and rigid lipid film, thus eliminating drug precipitation problems at the source of the process. Combined with ultrasonic hydration in a 37-40°C water bath, high-power pulsed ultrasonic homogenization, and gradient refinement processes, the parameters of each step are matched, and the material conversion is a closed-loop process. High-quality formulations can be stably produced in both small-scale and ten-fold scale-up experiments. Even after scale-up, the encapsulation efficiency remains at 95.2% and the drug loading is 12.1%. The process exhibits good repeatability and high tolerance, demonstrating excellent potential for industrial-scale production.

[0020] Furthermore, this invention employs a pulsed stepwise homogenization process, balancing the integrity of liposome formation with the uniformity of system dispersion. Combined with precision filtration using a ≤0.3μm sterile microporous membrane, it effectively removes large particles and impurities, ensuring the sterile and homogeneous injectable-grade quality of the formulation. Simultaneously, differentiated storage conditions are specifically designed: liquid formulations are stored at 2–8°C in the dark, while lyophilized formulations are stored at low temperatures for long-term preservation. This ensures stable quality throughout the entire process from production and storage to clinical use, effectively reducing the risk of formulation failure and deterioration.

[0021] This invention's liposome injection, leveraging the advantages of nanoparticle size, achieves targeted tumor accumulation through the penetration and retention effect in solid tumors, significantly shortening the treatment cycle while enhancing tumor suppression. In vivo animal efficacy studies show that, at the same dosage of 25 mg / kg, only five intravenous administrations every other day are needed, far exceeding the therapeutic effect of 21 days of continuous gavage administration of free baicalein. It demonstrates a clear inhibitory effect on four major solid tumors: breast cancer, ovarian cancer, lung cancer, and colorectal cancer, achieving a broad-spectrum anti-tumor therapeutic effect.

[0022] Furthermore, this invention quantitatively validates the advantages of differentiated treatment using multiple cancer animal models. The formulation exhibits a tumor inhibition rate of up to 77.3% against SKOV3 ovarian cancer, demonstrating optimal therapeutic efficacy; tumor inhibition rates of 55.3% and 52.2% against MC38 colorectal cancer and LLC lung cancer, respectively, showing stable tumor inhibition effects; and a tumor inhibition rate of 48.3% against 4T1 breast cancer, effectively delaying tumor proliferation. Based on the clearly defined quantitative differences in efficacy, this invention can be preferentially applied to targeted therapy for ovarian cancer, and is also suitable for adjuvant therapy for various solid tumors. It has strong clinical adaptability, high therapeutic targeting, and excellent clinical application value and drug development prospects. Attached Figure Description

[0023] Figure 1 Transmission electron microscopy (TEM) image (A), hydrated particle size (B), and zeta potential (C) of baicalin liposomes.

[0024] Figure 2 Images of baicalin liposomes prepared under different conditions.

[0025] Figure 3 The procedure for determining the encapsulation efficiency and drug loading of baicalin liposomes by high-speed centrifugation is as follows: (A) Standard curve established for the detection of baicalin standard by high-performance liquid chromatography; (B) Procedure for obtaining free and total baicalin by high-speed centrifugation; (C) Standard curve for lipid concentration determination by ferric ammonium thiocyanate colorimetric method.

[0026] Figure 4 The in vitro release curves of baicalin and baicalin liposome injection are shown.

[0027] Figure 5 The therapeutic effects of intravenous administration of baicalein liposomes on mouse models of breast cancer, lung cancer, colon cancer, and ovarian cancer are shown in Figure (A); the therapeutic effects of oral administration of baicalein on a mouse model of breast cancer are shown in Figure (B).

[0028] Figure 6 The in vivo toxicological results of healthy mice injected with physiological saline (control group) and baicalein liposome injection (drug treatment group) are shown in the figure. (A) Weight growth and differences between the two groups of mice; (B) Hematoxylin-eosin staining of the heart, liver, spleen, lungs and kidneys of the two groups of mice; (C) Blood routine levels and differences between the two groups of mice; (D) Blood biochemistry levels and differences in liver and kidney function between the two groups of mice. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0030] This invention provides a baicalein liposome injection, comprising two pharmaceutical dosage forms: a baicalein liposome formulation and a baicalein liposome lyophilized formulation. The baicalein liposome injection of this invention is an aqueous dispersion comprising liposomes, baicalein encapsulated in a hydrophobic phospholipid layer of the liposomes, and an aqueous medium. The prepared baicalein liposome injection has an average particle size of 50-150 nm, a PDI of 0.10-0.30, a baicalein encapsulation efficiency greater than or equal to 85%, and a baicalein drug loading greater than or equal to 10%. The final concentration of baicalein (active ingredient) in the injection is 4.5-7.5 mg / mL, and the aqueous medium is water for injection, such as physiological saline (0.9% sodium chloride) or 5% glucose solution.

[0031] In this invention, baicalin liposomes refer to a lipid bilayer structure composed of phospholipids and cholesterol, dispersed or suspended in an aqueous medium. This lipid bilayer is similar to a cell membrane structure, with baicalin embedded within the phospholipid bilayer. Baicalin liposome lyophilized formulations, in this invention, refer to lyophilized powder prepared by adding excipients to the baicalin liposome formulation and then freeze-drying it under vacuum. Before application, this lyophilized powder is reconstituted into liposome form by adding water for injection and shaking.

[0032] This invention provides a method for preparing a liposome formulation of baicalin, the specific steps of which are as follows: Step 1: Mix baicalein extract, egg yolk lecithin (phosphatidylcholine PC≥98%), and cholesterol in a weight ratio of 1:3:0.8~1.5, and add a solvent (such as chloroform or acetone) sufficient to fully dissolve the mixture to obtain mixture 1; Step 2: Place mixture 1 on a vacuum rotary evaporator to remove solvent under a vacuum of 0.08 MPa and a water bath at 37-40°C. After film formation, continue rotary evaporation for 15-35 minutes. Add water for injection (calculate the volume to be added based on the concentration of the active ingredient to be prepared). Perform ultrasonic hydration in a conventional ultrasonic cleaner at a water bath at 37-40°C. Then, place the mixture in a high-power non-contact ultrasonic cell disruptor for further ultrasonic homogenization to obtain mixture 2. Step 3: After filtering mixture 2 through a sterile microporous membrane with a micrometer or smaller than 0.3 micrometers, bottle it and store it at 2~8℃ in the dark to obtain a baicalin liposome dosage form.

[0033] This invention provides a method for preparing a lyophilized baicalein liposome dosage form, the specific steps of which are as follows: Step 1: Mix baicalein extract, egg yolk lecithin (phosphatidylcholine PC≥98%), and cholesterol in a weight ratio of 1:3:0.8~1.5, and add a solvent (such as chloroform or acetone) sufficient to fully dissolve the mixture to obtain mixture 1; Step 2: Place mixture 1 on a vacuum rotary evaporator to remove solvent under a vacuum of 0.08 MPa and a water bath at 37-40°C. After film formation, continue rotary evaporation for 15-35 minutes. Add a solution containing a freeze-drying protectant (3%-5% glucose concentration). Perform ultrasonic hydration in a conventional ultrasonic cleaner at a water bath at 37-40°C. Then, place the mixture in a high-power non-contact ultrasonic cell disruptor for further ultrasonic homogenization. Step 3: After filtering mixture 2 through a sterile microporous membrane with a pore size of 0.3 micrometers or less, bottle the mixture, freeze-dry it under sterile conditions, fill and seal it, and store it at 2-8°C protected from light. Before use, add water for injection to achieve a final concentration of baicalein (weight of baicalein (mg) / total volume of preparation) of 4.5-7.5 mg / mL.

[0034] It should be noted that the baicalin raw material used in this invention can be either commercially available baicalin extract or baicalin extract obtained independently from plants such as Scutellaria baicalensis, Scutellaria barbata, Scutellaria barbata, Oroxylum indicum, and Plantago asiatica; among them, extracts with a baicalin purity of not less than 98 wt% are preferred raw materials. Furthermore, the egg yolk lecithin, cholesterol, water for injection, and other excipients used in this invention all meet pharmaceutical standards, with injection-grade refined egg yolk lecithin being preferred, effectively ensuring the purity of the formulation and the safety of clinical use from the source of raw materials.

[0035] Example 1 This embodiment uses a single-factor variable experiment to screen and optimize key formulations and process parameters that affect the liposome formation effect, drug loading level and storage stability of baicalein. After determining the optimal preparation conditions, small-scale preparation verification and large-scale process scale-up verification under the optimal parameters are carried out.

[0036] First, a purity screening test of phospholipid raw materials was conducted. Liposomes were prepared using egg yolk lecithin (Shenyang Tianfeng Biopharmaceutical, EPC-80) with a phosphatidylcholine (PC) content ≥80% and egg yolk lecithin (Shenyang Tianfeng Biopharmaceutical, EPC-98) with a PC content ≥98%. The results showed that lecithin with only 80% PC content could not form a complete closed phospholipid bilayer structure in the aqueous phase, easily forming phospholipid aggregates, ultimately resulting in a flocculent turbid solution. However, when using egg yolk lecithin with a PC content ≥98%, a typical homogeneous liposome colloidal solution could be prepared, and no yellow baicalin solid precipitated. Figure 2 a). Therefore, it is determined that the preparation process of this invention requires the use of egg yolk lecithin with a PC content ≥98% as the membrane material raw material.

[0037] Secondly, screening experiments were conducted on the organic solvent removal process conditions. Two control processes were set up under vacuum ≥0.08 MPa and water bath film formation conditions of 37~40℃: In the first group, rotary evaporation was stopped immediately after lipid membrane formation and the absence of liquid organic solvent, and hydration was performed. The lipid membrane structure obtained by this method was loose, and yellow baicalin solid precipitated after the sample was allowed to stand. Figure 2 (b) In the second group, after the lipid membrane was formed, rotary evaporation was continued under a vacuum of 0.08 MPa or higher and a water bath temperature of 37–40°C for 15–35 minutes. This resulted in a hard and dense lipid film. After thorough hydration under high temperature and strong mechanical force, a homogeneous colloidal solution was formed without drug precipitation. Based on this, it was determined that the rotary evaporation time should be extended after film formation to completely remove trace amounts of organic solvent and avoid drug precipitation.

[0038] A second screening experiment was conducted to optimize the formulation. The weight ratio of baicalein to egg yolk lecithin was fixed at 1:3, and the cholesterol dosage was gradually adjusted to investigate its effect on the storage stability of liposomes. The results showed that when the relative cholesterol mass was 1.5–2.0, the prepared liposome colloidal solution exhibited significant precipitation after standing at 4°C for 24 hours; when the relative cholesterol mass was controlled within the range of 0.8–1.5, the resulting colloidal solution remained stable and homogeneous even after being sealed and stood at 4°C for 30 days. Figure 2c). Analysis shows that excessive cholesterol occupies drug loading sites in the hydrophobic cavities of the phospholipid membrane, disrupting membrane structural regularity, reducing encapsulation efficiency, and causing particle aggregation and sedimentation. Therefore, the preferred material weight ratio range for this invention is baicalein: egg yolk lecithin: cholesterol = 1:3:0.8~1.5.

[0039] Based on the selected raw material specifications, process conditions, and the determination of the ratio of baicalin:egg yolk lecithin:cholesterol = 1:3:1.2, a small-scale batch preparation verification was carried out. The specific operation steps for the preparation method of the above-mentioned baicalin liposome dosage form are as follows: Weigh 10 mg of baicalein (purity ≥98%), 30 mg of egg yolk lecithin (phosphatidylcholine PC ≥98%), and 12 mg of cholesterol in a weight ratio of 1:3:1.2 and mix them in a 100 mL square bottle. Add 2 mL of chloroform and 1 mL of methanol to fully dissolve the mixture. Place the mixture in a vacuum rotary evaporator and remove the solvent under a vacuum of 0.085 MPa and a 38°C water bath. After 10 minutes, a film will form and no liquid organic solvent will be present. Continue rotary evaporation for 20 minutes, add 1.5 mL of physiological saline for injection, and perform ultrasonic hydration at 37°C using a conventional ultrasonic cleaner. Transfer the mixture to centrifuge tubes and place them in a non-contact ultrasonic cell disruptor (model SCIENTZ08-IIIC, 1800W). Set the power to 50%, sonicate for 5 seconds, and then sonicate intermittently for 5 seconds under a water bath temperature below 10°C for 4 minutes. After filtration through a 0.2-micron sterile microporous membrane, bottle the mixture and store it at 2-8°C in the dark. No precipitation will occur after standing for 30 days.

[0040] Systematic characterization and testing of the samples prepared in the small-scale test showed the following results: The final concentration of baicalein (weight of baicalein (mg) / total volume of preparation) was determined by liquid chromatography to be 4.8 ± 0.1 mg / mL, with an encapsulation efficiency of 97.3% and a drug loading of 12.5%. No precipitation or stratification was observed after 30 days of standing. Transmission electron microscopy revealed that the formed particles were nanoparticles encapsulated in a phospholipid bilayer. Figure 1 A), the average hydrated particle size of DLS is 90.5 nm ( Figure 1 B), the potential in PBS is -10.5mV ( Figure 1 C), with good colloidal dispersibility.

[0041] To verify the repeatability and industrial applicability of the preparation process, a tenfold scale-up experiment was conducted. The specific steps of the above-mentioned method for preparing a baicalin liposome dosage form are as follows: Weigh 100 mg of Scutellaria baicalensis (purity ≥98%), 300 mg of egg yolk lecithin (phosphatidylcholine PC ≥98%), and 120 mg of cholesterol in a weight ratio of 1:3:1.2 and mix them in a 250 mL square bottle. Add 20 mL of chloroform and 10 mL of methanol to fully dissolve the mixture. Place the mixture in a vacuum rotary evaporator and remove the solvent under a vacuum of 0.085 MPa and a water bath at 38°C. After 20 minutes, a film will form and no liquid organic solvent will be present. Continue rotary evaporation for another 25 minutes. Add 15 mL of physiological saline for injection. After ultrasonic hydration at 37°C using a conventional ultrasonic cleaner, transfer the mixture to centrifuge tubes and place them in a non-contact ultrasonic cell disruptor (model SCIENTZ08-IIIC, 1800W). Set the power to 50%, sonicate for 5 seconds with 5 seconds of intermittent sonication, and further homogenize the mixture under a water bath at below 10°C for 5 minutes. Filter the mixture through a 0.2-micron sterile microporous membrane and bottle it. Store at 2-8°C in the dark.

[0042] Scale-up sample preparation and testing results showed that the final concentration of baicalein was 4.7±0.1 mg / mL, the encapsulation efficiency was 95.2%, the drug loading was 12.1%, and the system remained stable without abnormalities after 30 days of standing. Figure 2 d) This demonstrates that the process of the present invention has good scale-up stability and strong repeatability, and has the potential for large-scale production.

[0043] Furthermore, systematic quality characterization and in vitro drug release performance testing were conducted on the small-scale samples prepared in Example 1. In vivo antitumor efficacy and biosafety testing were performed using scaled-up samples, as detailed below: 1. Encapsulation efficiency and drug loading detection 1.1) The calculation formula is as follows: Encapsulation efficiency calculation formula = [(W 总 - W 游 ) / W 总 ] × 100% Formula for calculating drug loading = [(W 总 -W 游 ) / (W 总 -W 游 +W 脂 )] × 100% The steps for obtaining the relevant parameters are as follows: 1.2) Take 200 μL of baicalin liposome suspension, centrifuge at 15000g for 20 minutes, take 100 μL of supernatant and mix with 400 μL of methanol, then detect by high performance liquid chromatography, and add to the standard curve established with baicalin standard to obtain the amount of free baicalin W. 游 ( Figure 3 A, Figure 3 B(a)).

[0044] 1.3) Simultaneously, 200 μL of baicalin liposome suspension was mixed with 800 μL of methanol and placed in a non-contact ultrasonic cell disruptor (model SCIENTZ08-IIIC, 1800W). The mixture was set to 50% power, with an ultrasonic frequency of 5 seconds followed by 5 seconds of intermittent sonication at temperatures below 10°C for 4 minutes to disrupt the emulsion. The mixture was then centrifuged at 15000g for 20 minutes. The supernatant was collected and analyzed by high-performance liquid chromatography to obtain the total amount of baicalin (W). 总 ( Figure 3 B(b)).

[0045] 1.4) The chromatographic conditions for the detection of baicalin by high performance liquid chromatography are as follows: the mobile phase is (A) methanol and (B) 0.5% formic acid; the elution program is 0~10 min 40%~60% phase A, 10~40 min 40%~60% phase A, 40~60 min 60%~100% phase A; column temperature: 30ºC, detection wavelength: 275nm.

[0046] 1.5) Lipid content detection: 15 mg egg yolk lecithin (PC ≥ 98%) and 6 mg cholesterol were dissolved in chloroform to prepare a lipid standard stock solution. The lipid stock solution was diluted with chloroform to obtain lipid standard solutions with concentrations of 25, 50, 100, 200, and 400 μg / mL.

[0047] Take a baicalein liposome suspension, dilute it 100-fold with chloroform, and then mix 500 μL of the sample or lipid standard with 500 μL of ferric ammonium thiocyanate reagent (a mixture of 10 mM FeCl3 and 40 mM NH4SCN). After centrifugation with shaking for 1 minute, measure the absorbance at 475 nm of the chloroform phase. Substitute this absorbance into the standard curve established for the standard lipids to obtain the amount of lipid W. 脂 ( Figure 3 C).

[0048] 2. Evaluation of in vitro sustained-release performance Take 1 mL of baicalein solution (1 mg / mL dissolved in ethanol) and 1 mL of the baicalein liposomes of this invention (containing 1 mg of baicalein) respectively, place them in dialysis bags with a molecular weight of 8000~14000D, tie both ends tightly, and place them in a beaker containing 50 mL PBS (containing 0.25% Tween-80). Determine the in vitro drug release performance using the paddle plate method at a constant temperature of 37℃ (50 r / min). Take 500 μL samples from the beaker at time points of 0, 2, 6, 12, 24, and 36 hours, and simultaneously add an equal volume of release medium for HPLC detection. Calculate the cumulative release amount using the following formula: Cumulative release rate (%) = (cumulative amount of drug released / total amount of drug in the formulation) × 100%.

[0049] As attached Figure 4As shown, the release rate of free baicalein was faster than that of baicalein liposomes within 0-5 hours; after 12 hours, the cumulative release rate of baicalein liposomes gradually increased compared to that of free baicalein; finally, at 36 hours, the cumulative release rates of baicalein from free baicalein and baicalein liposomes were 56.6±4.7% and 67.0±5.0%, respectively. This indicates that baicalein liposome injection can delay the release of baicalein and prolong its therapeutic duration.

[0050] 3. Evaluation of the efficacy of broad-spectrum antitumor drugs in vivo for multiple cancer types To evaluate the broad-spectrum antitumor activity of the baicalin liposomes of this invention, SPF-grade female BALB / c mice and C57BL / 6 mice were used for in vivo tumor suppression experiments. All experimental mice were housed in an SPF-grade barrier environment with a temperature of 22–25 °C, a relative humidity of 50% ± 10%, and alternating light and dark conditions for 12 hours each time. They had free access to food and water and were used for model construction after one week of acclimatization.

[0051] Subcutaneous ectopic tumor models were used to construct animal models of 4T1 breast cancer, SKOV3 ovarian cancer, LLC lung cancer, and MC38 colorectal cancer. The 4T1 model was constructed using female BALB / c mice, the LLC and MC38 tumor models using female C57BL / 6 mice, and the SKOV3 tumor model using female BALB / c-nu mice. Tumor cells in the logarithmic growth phase were harvested, and after adjusting the cell suspension concentration, they were subcutaneously inoculated into the right back of mice at a uniform rate to establish the model. The tumors were induced when their subcutaneous volume reached 100–150 mm. 3 Once the mice had reached a stable weight, and the tumors grew uniformly without ulceration, they were randomly divided into groups for drug intervention experiments.

[0052] For intravenous administration, the saline injection group served as the control group, and the baicalein liposome injection group served as the treatment group. The dosage was uniformly set at 25 mg (baicalein) / kg (mice), administered via tail vein injection every other day, for a total of 5 administrations, with a total treatment period of 11 days. Figure 5 A).

[0053] To compare the therapeutic efficacy with that of free baicalein, a subset of 4T1 tumor model mice underwent gavage treatment with baicalein. The control group received 0.5% sodium carboxymethyl cellulose dispersible via gavage, while the treatment group received free baicalein (dispersed in 0.5% sodium carboxymethyl cellulose) via gavage. The medication was administered daily via gavage at a dose of 25 mg / kg for 21 consecutive days. Figure 5 B).

[0054] Mouse body weight and tumor volume were recorded every other day starting from the day of drug administration. At the end of the experimental period, mice were sacrificed, tumor tissue was removed and weighed, and the tumor inhibition rate for each group was calculated as shown in Table 1, evaluating the in vivo antitumor effect of the drug. The formula for calculating the tumor inhibition rate is as follows: Tumor inhibition rate % = [(tumor weight of untreated model - tumor weight of treated model) / tumor weight of untreated model] × 100%.

[0055] Table 1. Efficacy (tumor inhibition rate) of subcutaneous inoculation in different tumor models.

[0056] The tumor inhibition rate statistics showed that the baicalin liposomes of this invention exhibited tumor inhibition rates of 48.3%, 77.3%, 52.2%, and 55.3% against 4T1 breast cancer, SKOV3 ovarian cancer, LLC lung cancer, and MC38 colorectal cancer, respectively, demonstrating a clear broad-spectrum anti-tumor effect and differentiated advantages in inhibitory efficacy against different tumor cell types. Among these, the formulation showed the most significant inhibitory effect against SKOV3 ovarian cancer, with a tumor inhibition rate approaching 80%, indicating that this liposomal formulation can significantly enhance the targeted inhibitory ability of baicalin against solid tumors of ovarian cancer. The inhibitory effects on colorectal cancer and lung cancer were secondary, with tumor inhibition rates both exceeding 50%, effectively inhibiting the proliferation process of tumors in vivo. It also maintained a tumor inhibition effect of nearly 50% against breast cancer, effectively delaying tumor growth and preventing invasive tumor proliferation. Compared to the free baicalin gavage group, under the same dosage conditions, this invention, with only an 11-day short-cycle intravenous administration regimen, achieved a therapeutic effect far exceeding that of the free drug administered via long-term gavage for 21 days. These results fully demonstrate that the present invention, through liposome carrier encapsulation technology, significantly improves the enrichment efficiency and bioavailability of drugs in tumor tissues, greatly enhances anti-tumor efficacy, shortens the treatment cycle, and avoids the burden of body tolerance caused by long-term and frequent administration. It has significant dosage form and application advantages in the treatment of various solid tumors.

[0057] 4. Systematic evaluation of the safety of in vivo administration of the drug To evaluate the in vivo biocompatibility of the baicalin liposomes of this invention, SPF-grade healthy female BALB / c mice were used for a systematic safety evaluation. The mouse housing environment, temperature, humidity, and circadian rhythm conditions were kept consistent with those of tumor model mice to ensure standardization and uniformity of the experimental system. The dosing regimen was based on the effective therapeutic dose from the efficacy studies, with a dosage of 25 mg / kg, administered via tail vein injection every other day for a total of 5 doses. Control mice were injected with 100 μL of physiological saline at the same frequency. Throughout the experiment, the mice's weight changes, mental state, diet, and activity were dynamically monitored. After the dosing cycle, peripheral blood was collected from the mice to detect complete blood count and core biochemical indicators of liver and kidney function, evaluating hematological and organ toxicity. Finally, the heart, liver, spleen, lungs, and kidneys were dissected and removed. After routine fixation, gradient dehydration, paraffin embedding, and serial sectioning, the tissues were stained with hematoxylin and eosin (HE), and the pathological morphological changes of each organ tissue were observed under an optical microscope to determine whether the formulation caused organ toxicity and inflammatory damage.

[0058] The results showed that, compared with the control group, baicalin liposome injection had no significant effect on the weight gain of mice. Figure 6 A); No significant histopathological changes were observed in the major organs (including heart, liver, spleen, lungs, and kidneys) of the treated mice. Figure 6 B); Although the white blood cell count (WBC), lymphocyte count (Lym#), and neutrophil count (Neu#) were somewhat decreased, they were still within the normal range. Figure 6 C); Liver (ALT, AST, ALB, T-BIL) and kidney (UREA, CR) function indicators remained largely unchanged. Figure 6 D). This indicates that baicalein liposome injection has good safety under the experimental conditions.

[0059] The above animal-level antitumor therapeutic efficacy and safety test results demonstrate that this baicalin liposome injection with high encapsulation rate and drug loading has a promising clinical application prospect in antitumor therapy.

[0060] Example 2 This embodiment provides a method for preparing a lyophilized baicalein liposome dosage form, the specific steps of which are as follows: Weigh 10 mg of baicalein (purity ≥98%), 30 mg of egg yolk lecithin (phosphatidylcholine PC ≥98%), and 12 mg of cholesterol in a weight ratio of 1:3:1.2 and mix them in a 100 mL square flask. Add 2 mL of chloroform and 1 mL of methanol to fully dissolve the mixture. Place the flask on a vacuum rotary evaporator and remove the solvent under a vacuum of 0.085 MPa and a 38°C water bath. After 10 minutes, a film will form and no liquid organic solvent will be present. Continue rotary evaporation for 20 minutes. Add 1.5 mL of 5% glucose solution preheated to 37°C (excipient, to avoid over-freezing). After the liposomes were destroyed, they were ultrasonically hydrated at 37°C using a conventional ultrasonic cleaner, then transferred to centrifuge tubes and placed in a non-contact ultrasonic cell disruptor (model SCIENTZ08-IIIC, 1800W). The power was set to 50%, with ultrasonic treatment for 5 seconds followed by 5 seconds of intermittent sonication. The mixture was then further homogenized by ultrasonic treatment for 4 minutes in a water bath below 10°C. After filtration through a 0.2-micron sterile microporous membrane, the mixture was incubated at -80°C overnight and then freeze-dried in a vacuum freeze dryer to obtain freeze-dried baicalin liposomes, i.e., the basal liposome freeze-dried formulation. Figure 2 e). The lyophilized scutellaria baicalensis liposome dosage form can be stored for a long time in the dark at 4℃, -20℃, and -80℃. Add 1.5 mL of physiological saline for injection before use. The liposome precipitate was obtained by the above-mentioned high-speed centrifugation method. After demulsification with methanol, the concentration of baicalein was determined by HPLC to be 4.5 ± 0.2 mg / mL. The encapsulation efficiency was 95.6%, the drug loading was 12.3%, and the liposome particle size and dispersibility showed no significant changes. All performance indicators met the standards for injectable formulations.

Claims

1. A baicalin liposome, characterized in that, The formulation includes phospholipid bilayer liposome vesicles and baicalin embedded in the hydrophobic layer of the liposome phospholipid layer; the phospholipid bilayer liposome vesicle membrane material includes egg yolk lecithin and cholesterol; the egg yolk lecithin contains ≥98% phosphatidylcholine (PC) by mass; the baicalin liposomes have an average hydrated particle size of 50~150 nm, a PDI of 0.10~0.30, a baicalin encapsulation efficiency of ≥85%, and a baicalin drug loading of ≥10%.

2. A lyophilized baicalin liposome formulation, characterized in that, The baicalein liposome lyophilized formulation is prepared by vacuum freeze-drying of the baicalein liposomes as described in claim 1 and a lyophilization excipient.

3. The basalpinx liposome lyophilized formulation according to claim 2, characterized in that, The lyophilization excipient is glucose with a concentration of 3% to 5%.

4. A baicalin liposome injection, characterized in that, The baicalein liposome injection is prepared by mixing the baicalein liposome of claim 1 or the baicalein liposome lyophilized preparation of claim 2 with an aqueous injection medium, wherein the final concentration of baicalein in the preparation is 4.5~7.5 mg / mL.

5. The method for preparing a baicalin liposome injection according to claim 4, characterized in that, Includes the following steps: Baicalein, egg yolk lecithin, and cholesterol are mixed at a weight ratio of 1:3:0.8~1.5, and then dissolved completely in an organic solvent to obtain a mixture solution. The mixture solution was subjected to vacuum rotary evaporation, with the rotary evaporation vacuum degree controlled at ≥0.08MPa and the water bath temperature at 37~40℃. After the lipid film was formed, the rotary evaporation was continued for 15~35min under the same conditions to obtain a dense lipid film. When preparing liquid injection dosage forms, an aqueous medium for injection is added to the lipid film, followed by hydration and homogenization treatment to obtain a uniformly dispersed crude dispersion of liposomes. When preparing lyophilized injection formulations, a glucose aqueous solution with a mass concentration of 3% to 5% is added to the lipid film, followed by hydration and homogenization treatment to obtain a uniformly dispersed crude dispersion of liposomes. The obtained crude liposome dispersion was filtered through a sterile microporous membrane for sterilization. After filtration in liquid form, baicalein liposome injection was obtained. After filtration in lyophilized form, it was subjected to vacuum freeze-drying to obtain lyophilized baicalein liposome preparation. The lyophilized baicalein liposome preparation was mixed with an aqueous medium for injection and shaken to obtain lyophilized baicalein liposome injection.

6. The method for preparing a baicalin liposome injection according to claim 5, characterized in that, The hydration and homogenization process is as follows: First, the lipid film is hydrated using a conventional ultrasonic cleaner in a water bath at 37~40℃. Then, a non-contact ultrasonic cell disruptor is used for pulse homogenization. The ultrasonic working mode is 900W, with 5 seconds of ultrasonic treatment followed by 5 seconds of intermittent treatment.

7. The method for preparing a baicalin liposome injection according to claim 5, characterized in that, The filtration and sterilization process uses a sterile microporous filter membrane with a pore size ≤0.3μm.

8. The method for preparing a baicalin liposome injection according to claim 5, characterized in that, The storage conditions for the finished product are as follows: liquid dosage forms should be stored at 2~8℃ away from light, and lyophilized dosage forms should be stored at 4℃~-80℃ in a sealed container away from light.

9. The use of the baicalin liposome injection according to claim 4 in the preparation of a broad-spectrum antitumor drug, characterized in that, The antitumor drug is an intravenously administered preparation, and the types of tumors it is suitable for treating include breast cancer, ovarian cancer, lung cancer, and colorectal cancer.

10. The application according to claim 9, characterized in that, The baicalin liposome injection is used to prepare an anti-ovarian cancer drug.

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