Methylene blue liposome compositions and methods for their preparation
Patent Information
- Application Number
- CN202510348197.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-24
- Publication Date
- 2026-09-25
AI Technical Summary
但是，采用乙醚注入法得到的亚甲蓝脂质体，其包封率低，稳定性也需要进一步提高
[0023]本发明亚甲蓝脂质体，采用特定的主动载药法制备得到，其包封率高，包载效率高，成本低，安全性好，易于工业化大生产。
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Figure CN122805576A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to methylene blue liposome compositions and their preparation methods, belonging to the field of pharmaceutical formulation technology. Background Technology
[0002] Methylene blue, chemical formula C 16 H 18 N3ClS is a phenothiazine salt, a dark green bronze-lustered crystal or powder, soluble in water and ethanol, but insoluble in ethers. Methylene blue is relatively stable in air, and its aqueous solution is alkaline. The molecular structure of methylene blue is:
[0003]
[0004] Methylene blue is a dye compound. Industrial-grade methylene blue is frequently used for dyeing cotton, silk, and paper, and can also be used for coloring bamboo and wood, and in the manufacture of inks and lakes. Additionally, it is commonly used as a bacterial stain and indicator. In the medical field, methylene blue has long been used.
[0005] As early as the 1890s, methylene blue was successfully used to treat bacterial malaria, marking the first instance of using a synthetically produced dye compound to treat a disease. Research into dye-based disease treatment has continued ever since. Due to its redox properties, methylene blue can be used to treat poisoning caused by cyanide, nitrite, aniline, acetanilide, or methemoglobinemia induced by sulfonamides and other drugs. In recent years, researchers have also extensively studied the applications of methylene blue and its metabolites in various bacterial and viral infections, cancer, and central nervous system diseases such as depression, schizophrenia, Alzheimer's disease, and preoperative surgical tracking. However, methylene blue is also a toxic substance. Ordinary methylene blue injections require large doses and are prone to diffusion, posing certain health risks to the human body, such as blue urine, itchy skin, dizziness, headache, and difficulty breathing. Therefore, it is particularly necessary to develop safer and more effective dosage forms to improve the bioavailability and therapeutic or labeling effects of methylene blue, while reducing the dosage and ensuring the safety of methylene blue administration, in order to ensure its effectiveness, safety, and reliability in clinical applications.
[0006] Liposomes are drug carriers with advantages such as good stability, targeted drug delivery, membrane structure, drug carrying capacity, and biocompatibility, and can be used for the delivery of various drugs. Currently, due to the strong hydrophilicity of methylene blue, liposomes have been prepared using the ether injection method. However, the encapsulation efficiency of methylene blue liposomes obtained by the ether injection method is low, and their stability needs further improvement. There are currently no reports on active drug delivery systems for methylene blue liposomes with high encapsulation efficiency and high drug loading capacity (such as the ammonium sulfate gradient method). Therefore, this invention attempts to develop a stable methylene blue liposome composition with high encapsulation efficiency and high drug loading capacity, as well as its active drug delivery preparation method, to better utilize the pharmaceutical applications of methylene blue liposomes. Summary of the Invention
[0007] To address the above deficiencies, the technical problem solved by this invention is to provide a methylene blue liposome composition with high encapsulation efficiency and high drug loading rate, and a method for preparing the same.
[0008] The present invention relates to a methylene blue liposome composition comprising methylene blue and a lipid material, wherein the lipid material is a neutral phospholipid and cholesterol, and the weight ratio of cholesterol to neutral phospholipid is 1:1 to 100, and the weight ratio of methylene blue to lipid material is 1:5 to 100, wherein the neutral phospholipid is hydrogenated soybean phospholipid or distearate phosphatidylcholine.
[0009] In one embodiment of the present invention, the weight ratio of methylene blue to lipid material is 1:10 to 30.
[0010] In one embodiment of the present invention, the average particle size of the methylene blue liposomes is 60–300 nm.
[0011] In one embodiment of the invention, the methylene blue liposome composition further includes distearate phosphatidylethanolamine-polyethylene glycol 2000.
[0012] In one embodiment of the present invention, the weight ratio of distearate phosphatidylethanolamine-polyethylene glycol 2000 to cholesterol is 0.01 to 1:1.
[0013] In one embodiment of the present invention, the methylene blue liposomes have an encapsulation efficiency >90%, a loading efficiency >90%, and a stability time ≥5 days when incubated in 5% human serum albumin simulated plasma at 37°C.
[0014] The present invention also provides a method for preparing a methylene blue liposome composition.
[0015] The method for preparing the methylene blue liposome composition of the present invention includes the following steps:
[0016] a. Preparation of blank liposomes: Neutral phospholipids and cholesterol are dissolved in anhydrous ethanol to obtain an organic phase; the organic phase is injected into an aqueous solution, and the liposomes are processed to 60-300 nm through one-step emulsification or homogenization emulsification, microfluidization or extrusion process to obtain blank liposomes; the aqueous solution is an ammonium sulfate solution or a buffer salt solution with a pH of 4.0.
[0017] b. Constructing a gradient: Prepare a buffer salt solution with a pH of 5.0 to 7.4 as a replacement medium to replace the in vitro aqueous phase of the blank lipids, so that the difference in ammonium ion gradient between the in vitro and in vivo aqueous phases of the lipids is at least 2000 times or the difference in pH between the in vitro and in vivo aqueous phases of the lipids is at least 3 gradients.
[0018] c. Drug loading: Add methylene blue solution to blank liposomes with a gradient difference and incubate for 5-30 min to obtain methylene blue liposomes.
[0019] In one embodiment of the present invention, in step a, the dissolution temperature is 50-70°C and the temperature of the aqueous solution is 50-70°C; in step c, the incubation temperature is 55-80°C.
[0020] In some specific embodiments of the present invention, in step b, the difference in ammonium ion gradient between the in vitro and in vivo aqueous phases of the lipid body is 2000 to 10000 times.
[0021] In one embodiment of the present invention, in step c, methylene blue and distearate phosphatidylethanolamine-polyethylene glycol 2000 solution are added together to blank liposomes having a gradient difference.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] The methylene blue liposomes of this invention are prepared using a specific active drug delivery method, and have high encapsulation efficiency, high loading efficiency, low cost, good safety, and are easy to mass-produce industrially.
[0024] The methylene blue liposomes of this invention have good stability and can be used in various bacterial and viral infections, cancer, and central nervous system diseases such as depression, schizophrenia, Alzheimer's disease, and preoperative tracking and labeling. Attached Figure Description
[0025] Figure 1 The particle size distribution of the methylene blue liposome injection solution in Formulation 1 of Example 1 is shown.
[0026] Figure 2 This is a product image of methylene blue liposome injection solution of formulation 1 in Example 1. Detailed Implementation
[0027] The present invention relates to a methylene blue liposome composition comprising methylene blue and a lipid material, wherein the lipid material is a neutral phospholipid and cholesterol, and the weight ratio of cholesterol to neutral phospholipid is 1:1 to 100, and the weight ratio of methylene blue to lipid material is 1:5 to 100, wherein the neutral phospholipid is hydrogenated soybean phospholipid or distearate phosphatidylcholine.
[0028] The methylene blue liposomes of this invention are prepared by the above method. The average particle size of these liposomes ranges from 60 to 300 nm. The developed liposomes exhibit good stability, high encapsulation efficiency, high loading efficiency, low cost, and good safety. They can be used in various bacterial and viral infections, cancer, and central nervous system diseases such as depression, schizophrenia, Alzheimer's disease, and preoperative tracking and labeling.
[0029] In one embodiment of the present invention, the weight ratio of methylene blue to lipid materials (neutral phospholipids and cholesterol) is 1:10 to 30.
[0030] In one embodiment of the present invention, the average particle size of the methylene blue liposomes is 60–300 nm.
[0031] In one embodiment of the invention, the methylene blue liposome composition further includes distearate phosphatidylethanolamine-polyethylene glycol 2000.
[0032] In one embodiment of the present invention, the weight ratio of distearate phosphatidylethanolamine-polyethylene glycol 2000 to cholesterol is 0.01 to 1:1.
[0033] In one embodiment of the present invention, the methylene blue liposomes have an encapsulation efficiency >90%, a loading efficiency >90%, and a stability time ≥5 days when incubated in 5% human serum albumin simulated plasma at 37°C.
[0034] The method for preparing the methylene blue liposome composition of the present invention includes the following steps:
[0035] a. Preparation of blank liposomes: Neutral phospholipids and cholesterol are dissolved in anhydrous ethanol to obtain an organic phase; the organic phase is injected into an aqueous solution, and the liposomes are processed to 60-300 nm through one-step emulsification or homogenization emulsification, microfluidization or extrusion process to obtain blank liposomes; the aqueous solution is an ammonium sulfate solution or a buffer salt solution with a pH of 4.0.
[0036] b. Constructing a gradient: Prepare a buffer salt solution with a pH of 5.0 to 7.4 as a replacement medium to replace the in vitro aqueous phase of the blank lipids, so that the difference in ammonium ion gradient between the in vitro and in vivo aqueous phases of the lipids is at least 2000 times or the difference in pH between the in vitro and in vivo aqueous phases of the lipids is at least 3 gradients.
[0037] c. Drug loading: Add methylene blue solution to blank liposomes with a gradient difference and incubate for 5-30 min to obtain methylene blue liposomes.
[0038] The method for preparing methylene blue liposome composition of the present invention is an active drug delivery method. It uses a pH gradient method or an ammonium ion gradient method to prepare methylene blue into liposomes, which has good stability, high encapsulation efficiency, low cost, good safety, and is easy to industrialize.
[0039] Step a is to prepare blank liposomes. Neutral phospholipids and cholesterol are dissolved in anhydrous ethanol to obtain an organic phase; the organic phase is injected into an aqueous solution, and the liposomes are processed to 60-300 nm using a one-step emulsification or homogenization emulsification, microfluidization or extrusion process to obtain blank liposomes with an average particle size range of 60-300 nm.
[0040] In one embodiment of the present invention, in step a, the dissolution temperature is 50-70°C, and the temperature of the aqueous solution is 50-70°C.
[0041] Step b involves constructing a concentration gradient by preparing a buffer solution with a pH of 5.0–7.4 as the replacement medium to replace the in vitro aqueous phase of the blank lipids, ensuring that the ammonium ion gradient difference between the in vitro and in vivo aqueous phases is at least 2000 times, or that a pH gradient difference of at least three levels is formed between the in vitro and in vivo aqueous phases. When ammonium sulfate is used as the aqueous phase solution in step a, an ammonium ion concentration gradient is constructed; when a buffer solution with a pH of 4.0 is used as the aqueous phase solution in step a, a pH gradient is constructed.
[0042] Step b is crucial in the preparation process. Establishing an appropriate ammonium ion transmembrane gradient ensures liposome loading efficiency, improves encapsulation rate, and enhances stability. In some specific embodiments of this invention, step b involves setting the ammonium ion gradient difference between the in vitro and in vitro aqueous phases of the liposomes to 2000–10000 times.
[0043] Step c is drug loading, where methylene blue is added to blank liposomes with a gradient difference and incubated for 5–30 min to obtain methylene blue liposomes.
[0044] The incubation temperature in step c also affects the active drug loading process of liposomes. When the incubation temperature increases, the drug loading rate increases, the drug loading time shortens, and the liposomes can reach drug loading equilibrium more quickly. However, when the temperature is too high, it will cause degradation of liposome phospholipids and drug, and increase impurities. In one embodiment of the present invention, the incubation temperature is 55-80°C, which can improve the encapsulation efficiency and encapsulation rate.
[0045] In one embodiment of the present invention, in step c, methylene blue and a distearylphosphatidylethanolamine-polyethylene glycol 2000 solution are jointly added to blank liposomes with a gradient difference. By adding distearylphosphatidylethanolamine-polyethylene glycol 2000 to prolong the methylene blue liposome labeling time, its stability can be further improved.
[0046] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.
[0047] Example 1: Preparation of methylene blue liposomes by ammonium ion gradient method
[0048] Prepare methylene blue liposomes according to the formulation in Table 1.
[0049] Table 1
[0050] formula Prescription 1 Prescription 2 Prescription 3 Prescription 4 Prescription 5 Methylene Blue (g) 0.5 0.5 1 1 1 DSPC(g) 7.5 / 7.5 10 10 HSPC(g) / 7.5 / / / Cholesterol (g) 2.5 2.5 2.5 1 0.1 PEG2000-DSPE(g) / / 0.5 1 0.1 Ammonium sulfate (g) 13.2 13.2 13.2 13.2 13.2 Phosphate-buffered saline (PBS) 100mM 100mM 100mM 100mM 100mM Add water for injection to a final volume of (mL). 1000 1000 1000 1000 1000
[0051] Among them, DSPC is distearylphosphatidylcholine, HSPC is hydrogenated soybean lecithin, and PEG2000-DSPE is distearylphosphatidylethanolamine-polyethylene glycol 2000.
[0052] Preparation method:
[0053] 1) Preparation of blank liposome colostrum: Dissolve the phospholipids and cholesterol in the prescribed amounts in Table 1 in anhydrous ethanol to obtain an organic phase and keep it at 65°C for storage; prepare a 200mM ammonium sulfate solution using water for injection at 65°C to obtain an aqueous phase and keep it at 65°C; inject the phospholipid ethanol organic phase solution into the ammonium sulfate aqueous phase; and process the liposomes to 60-300nm by extrusion.
[0054] 2) Construction of ammonium ion gradient in blank liposomes: A pH 6.5 buffer solution was prepared as the replacement medium, and the aqueous phase of the liposome was replaced by ultrafiltration to create a 2000-fold gradient difference of ammonium ions between the in vitro and in vitro aqueous phases.
[0055] 3) Drug loading: Prepare a solution of methylene blue and distearate phosphatidylethanolamine-polyethylene glycol 2000 (PEG2000-DSPE) and add it to a blank liposome with an ammonium ion gradient. Incubate at 70°C for 15 min to obtain a methylene blue liposome solution. After sterilization by filtration through a 0.22 μm filter membrane, fill the solution into vials to obtain a methylene blue liposome injection solution.
[0056] The methylene blue liposome injection solutions prepared according to each prescription were tested for content, particle size, and encapsulation efficiency. At the same time, particle stability was tested by incubation with 37℃ 5% human serum albumin simulated plasma. The results of quality testing and particle stability testing are shown in Table 2.
[0057] Table 2
[0058] prescription Prescription 1 Prescription 2 Prescription 3 Prescription 4 Prescription 5 Methylene blue content (mg / mL) 0.498 0.479 0.991 0.986 0.993 Particle size (nm) 148 132 138 122 128 Encapsulation efficiency (%) 99.5 95.8 99.1 98.6 99.3 Packing efficiency (%) 99.5 95.8 99.1 98.6 99.3 Stability time in simulated plasma (days) 5.5 5.0 6.0 6.5 7.5
[0059] The method for determining the methylene blue content is as follows, referring to the pharmacopoeia method: Accurately measure an appropriate amount of this product (approximately equivalent to 20 mg of methylene blue), quantitatively dilute with dilute ethanol to prepare a solution containing approximately 2 μg per mL. Measure the absorbance at a wavelength of 661 nm using ultraviolet-visible spectrophotometry (General Rule 0401). Separately, accurately weigh an appropriate amount of methylene blue reference standard, dissolve it in dilute ethanol, and quantitatively dilute to prepare a solution containing approximately 2 μg per mL. Measure the absorbance using the same method. Calculate the content of C in the test amount. 16 H 18 The amount of ClN3S·3H2O.
[0060] The particle size was determined using a Malvern Nano S90 laser nanoparticle size analyzer. Methylene blue liposomes were diluted to a suitable concentration and poured into a cuvette for direct particle size measurement, and the average particle size was recorded.
[0061] The encapsulation efficiency was determined by separating the liposome product using a membrane centrifuge tube. The free methylene blue solution after centrifugation was then analyzed to determine the methylene blue content, which is the free drug content. The encapsulation efficiency was calculated using the following formula.
[0062]
[0063] The encapsulation efficiency is determined by measuring the encapsulation ratio of a freshly loaded drug solution; at this point, the encapsulation ratio equals the encapsulation efficiency. The encapsulation efficiency is then calculated using the following formula.
[0064]
[0065] Method for determining the stability time in simulated plasma: Dilute 20% human serum albumin injection to 5% human serum albumin with pH 7.4 PBS buffer to prepare simulated plasma. Then, take 50 mL of 5% human serum albumin simulated plasma and place it in a polyethylene bottle, incubating it in a water bath at 37°C to simulate the human plasma environment. Next, add 1 mL of methylene blue liposome solution to the simulated plasma for incubation. Measurements are taken at 0 days, 0.5 days, 1 day, 1.5 days, 2 days, 2.5 days, 3 days… for particle size analysis. If the particle size is not less than 80 nm, continue analysis. If the particle size is greater than 80 nm at 4.5 days but less than 80 nm at 5 days, the stability time in the simulated plasma is 4.5 days.
[0066] Example 2: Preparation of methylene blue liposome injection solution using pH gradient method
[0067] Methylene blue liposome injection solution was prepared according to the following method:
[0068] 1) Preparation of blank liposome colostrum: Dissolve 7.5g DSPC and 2.5g cholesterol in anhydrous ethanol to obtain an organic phase and store it at 65℃; prepare a pH 4.0 100mM citrate buffer solution using water for injection at 65℃ to obtain an aqueous phase and keep it at 65℃; inject the phospholipid ethanol organic phase solution into the citrate buffer solution aqueous phase; process the liposomes to 60-300nm by homogenization emulsification, microfluidization, or extrusion.
[0069] 2) pH gradient construction of blank liposomes: 7.4 phosphate buffer solution was prepared as the replacement medium, and the aqueous phase of the liposome was replaced by ultrafiltration to form a pH gradient between the aqueous phase inside and outside the liposome. There are 3 pH gradients.
[0070] 3) Drug loading: Prepare 0.5g of methylene blue and 0.5g of distearylphosphatidylethanolamine-polyethylene glycol 2000 aqueous solution and add them to blank liposomes with a pH gradient. Incubate at 70℃ for 20min to obtain methylene blue liposome solution. After sterilization filtration through a 0.22μm filter membrane, fill into vials to obtain methylene blue liposome injection solution.
[0071] The prepared methylene blue liposome injection solution was tested for content, particle size, and encapsulation efficiency. At the same time, stability was tested using 5% human serum albumin simulated plasma. The experimental results showed that the methylene blue content of the liposomes prepared by the pH gradient method was 0.486 mg / mL, the encapsulation efficiency was 97.2%, the particle size was 143 nm, and it was stable for 6.0 days in 5% human serum albumin simulated plasma.
[0072] Comparative Example 1
[0073] The formulation of Formula 1 in Example 1 and the preparation method of Example 1 were used. The only difference was that in step 2), when constructing the ammonium ion gradient by ultrafiltration, the gradient difference of ammonium ions in the aqueous phase inside and outside the liposome was 1000 times, resulting in methylene blue liposome injection.
[0074] The prepared methylene blue liposome injection solution was tested for content, particle size, and encapsulation efficiency. At the same time, stability was tested using 5% human serum albumin simulated plasma. The experimental results showed that the methylene blue content of the liposome was 0.4 mg / mL, the encapsulation efficiency was 80.3%, the loading efficiency was 80%, the particle size was 153 nm, and it was stable for 5.0 days in 5% human serum albumin simulated plasma.
[0075] Comparative Example 2
[0076] Using the formulation of Formula 1 in Example 1 and the preparation method of Example 1, the only difference is that egg yolk lecithin is used instead of DSPC in the formulation to obtain methylene blue liposome injection.
[0077] The prepared methylene blue liposome injection solution was tested for content, particle size, and encapsulation efficiency. At the same time, stability was tested using 5% human serum albumin simulated plasma. The experimental results showed that the methylene blue content of the liposome was 0.43 mg / mL, the encapsulation efficiency was 86.2%, the loading efficiency was 86%, the particle size was 137 nm, and it was stable in 5% human serum albumin simulated plasma for 3.5 days.
[0078] Comparative Example 3
[0079] Methylene blue liposomes were prepared using the ether injection method, with the formulation being Formulation 1 of Example 1. The specific steps are as follows:
[0080] (1) Weigh methylene blue into PBS phosphate solution and stir in a 60°C water bath.
[0081] (2) Weigh DSPC for injection and cholesterol into a beaker and dissolve in 10 mL of ether;
[0082] (3) Slowly add this ether solution dropwise to a 60°C methylene blue solution, and then remove the ether by rotary evaporation to obtain a liposome suspension.
[0083] (4) The obtained suspension is processed a second time by a high-pressure homogenizer to obtain single-compartment liposomes.
[0084] (5) Then ultrafiltration is used to remove the unencapsulated methylene blue solution, followed by washing and concentration to obtain the final product.
[0085] The prepared methylene blue liposomes were tested for content, particle size, and encapsulation efficiency. Stability was also tested using 5% human serum albumin-simulated plasma. The results showed that the liposomes had a methylene blue content of 0.160 mg / mL, a particle size of 141 nm, a methylene blue encapsulation efficiency of 30%, and a stability time of 4.5 days in simulated plasma.
[0086] As can be seen from the above embodiments and comparative examples, the methylene blue liposomes prepared by the active drug loading method of the present invention have high encapsulation efficiency and high loading rate. Moreover, the methylene blue liposomes prepared by the formulation of the present invention have a stability of at least 5 days when incubated in simulated plasma, which can meet the requirements for surgical marking and long-term circulation in vivo to exert therapeutic effects.
Claims
1. A methylene blue liposome composition, characterized in that: The mixture includes methylene blue and lipid materials, wherein the lipid materials are neutral phospholipids and cholesterol, and the weight ratio of cholesterol to neutral phospholipids is 1:1 to 100, and the weight ratio of methylene blue to lipid materials is 1:5 to 100, wherein the neutral phospholipids are hydrogenated soybean phospholipids or distearate phosphatidylcholine.
2. The methylene blue liposome composition according to claim 1, characterized in that: The weight ratio of methylene blue to lipid material is 1:10 to 30.
3. The methylene blue liposome composition according to claim 1, characterized in that: The average particle size of methylene blue liposomes is 60–300 nm.
4. The methylene blue liposome composition according to claim 1, characterized in that: The methylene blue liposome composition also includes distearate phosphatidylethanolamine-polyethylene glycol 2000.
5. The methylene blue liposome composition according to claim 1, characterized in that: The weight ratio of distearate phosphatidylethanolamine-polyethylene glycol 2000 to cholesterol is 0.01 to 1:
1.
6. The methylene blue liposome composition according to claim 1, characterized in that: The encapsulation efficiency of methylene blue liposomes is >90%, the loading efficiency is >90%, and the stability time after incubation in 5% human serum albumin simulated plasma at 37℃ is ≥5 days.
7. A method for preparing the methylene blue liposome composition according to any one of claims 1 to 6, characterized in that, Includes the following steps: a. Preparation of blank liposomes: Neutral phospholipids and cholesterol are dissolved in anhydrous ethanol to obtain an organic phase; the organic phase is injected into an aqueous solution, and the liposomes are processed to 60-300 nm through one-step emulsification or homogenization emulsification, microfluidization or extrusion process to obtain blank liposomes; the aqueous solution is an ammonium sulfate solution or a buffer salt solution with a pH of 4.
0. b. Constructing a gradient: Prepare a buffer salt solution with a pH of 5.0 to 7.4 as a replacement medium to replace the in vitro aqueous phase of the blank lipids, so that the difference in ammonium ion gradient between the in vitro and in vivo aqueous phases of the lipids is at least 2000 times or the difference in pH between the in vitro and in vivo aqueous phases of the lipids is at least 3 gradients. c. Drug loading: Add methylene blue solution to blank liposomes with a gradient difference and incubate for 5-30 min to obtain methylene blue liposomes.
8. The method for preparing the methylene blue liposome composition according to claim 7, characterized in that: In step a, the dissolution temperature is 50–70°C, and the temperature of the aqueous solution is 50–70°C; in step c, the incubation temperature is 55–80°C.
9. The method for preparing the methylene blue liposome composition according to claim 6, characterized in that: In step b, the difference in ammonium ion gradient between the in vitro and in vivo aqueous phases of the lipid cell is set to 2000–10000 times.
10. The method for preparing the methylene blue liposome composition according to claim 6, characterized in that: In step c, methylene blue and distearate phosphatidylethanolamine-polyethylene glycol 2000 solution are added together to blank liposomes with a gradient difference.