Tracer liposome solution and preparation method thereof

Abstract

The invention discloses a tracer liposome solution and a preparation method thereof. The tracer liposome comprises a carrier liposome and a tracer aqueous solution encapsulated in the carrier liposome, the carrier liposome comprises phospholipid, cholesterol and an encapsulating agent; the phospholipid comprises one or more of distearoyl phosphatidylcholine, hydrogenated soybean phospholipid and sphingomyelin; the tracer agent is one or more of methylene blue, indocyanine green and mitoxantrone. The tracer liposome solution not only is high in clinical use identification degree, small in clinical local stimulation and fast in local dissipation after being used, but also has high drug loading capacity.

Description

Technical Field

[0001] The present invention relates to a tracer liposome solution and a preparation method thereof. Background Art

[0002] In clinical use, the methylene blue preparation is methylene blue injection. During its clinical use, there will be problems such as relatively large local irritation and pain in patients. In addition, the dissipation time of methylene blue at the injection site is very long, affecting the normal life of patients. Currently, the tracers clinically used also include nano-carbon suspension injection and mitoxantrone hydrochloride, and both also have the defect of relatively large local irritation during clinical use.

[0003] Although there are examples of preparing tracers into liposomes in the prior art, the problem of poor drug loading needs to be solved urgently. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the problems in the prior art that the commercially available lymph node tracer varieties have relatively large irritation during clinical use, or the drug loading needs to be improved urgently, and provide a tracer liposome solution and a preparation method thereof. The tracer liposome solution of the present invention not only has high clinical recognition, small local irritation during clinical use, and relatively fast local dissipation after use, but also can have a high drug loading.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The present invention provides a tracer liposome solution, which includes carrier liposomes and a tracer aqueous solution encapsulated in the carrier liposomes;

[0007] Among them, the carrier liposomes include phospholipids, cholesterol, and encapsulating agents; the phospholipids include one or more of distearoyl phosphatidylcholine, hydrogenated soy phospholipid, and sphingomyelin; the tracer is one or more of methylene blue, indocyanine green, and mitoxantrone.

[0008] In the present invention, in the tracer liposome solution, the phospholipids and the cholesterol form a bilayer phospholipid molecular layer, and the bilayer phospholipid molecular layer and the encapsulating agent together form the carrier liposomes.

[0009] In the present invention, the carrier liposomes are generally the same as the blank liposome solution described in step S2 of the following preparation method.

[0010] In the present invention, the carrier liposomes generally also contain water. The water is generally introduced when dissolving the encapsulating agent in the preparation method.

[0011] In the present invention, the phospholipids are preferably distearoyl phosphatidylcholine and / or hydrogenated soy phospholipid.

[0012] In the present invention, the mass ratio of the phospholipid to the cholesterol may be (1 - 10):1, preferably (2 - 9):1, such as 3:1, 4:1, 5:1 or 7:1.

[0013] In the present invention, the type of the encapsulating agent may be ammonium sulfate and / or sucrose octasulfate, preferably ammonium sulfate.

[0014] In the present invention, the tracer may be methylene blue, indocyanine green or mitoxantrone, preferably methylene blue.

[0015] In the present invention, the molar ratio of the tracer to the encapsulating agent may be ≥(1:1), preferably ≥(1.5:1), such as 2.0:1, 2.5:1 or 3:1.

[0016] In the present invention, the water in the aqueous solution of the tracer is generally water for injection.

[0017] In the present invention, in the liposome solution of the tracer, the particle size of the liposome may not be less than 70 nm, preferably 70 nm - 200 nm, more preferably 80 nm - 200 nm, such as 90 nm, 100 nm, 131 nm, 132 nm, 133 nm, 134 nm, 150 nm or 180 nm.

[0018] In the present invention, in the liposome solution of the tracer, the drug-lipid ratio may be ≥36%, preferably ≥42%, such as 42%, 44%, 45%, 47%, 48%, 52%, 53%, 58%, 59%, 60%, 61%, 63%, 64%, 67%, 69%, 70%, 71%, 72%, 75%, 76%, 77%, 78%, 79%, 80% or 81%. The drug-lipid ratio generally refers to the ratio of the tracer concentration (mg / mL) to the phospholipid concentration (mg / mL) in the liposome solution of the tracer.

[0019] In some preferred embodiments, in the liposome solution of the tracer, the drug-lipid ratio may be in the range of 42% - 81%.

[0020] In the present invention, in the tracer liposome solution, the content of LPC can be < 1 mg / mL, preferably < 0.76 mg / mL, such as 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.10 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.18 mg / mL, 0.21 mg / mL, 0.26 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.37 mg / mL, 0.40 mg / mL, 0.48 mg / mL, 0.5 mg / mL, 0.57 mg / mL or 0.76 mg / mL.

[0021] In the present invention, in the tracer liposome solution, the total impurity content of related substances can be ≤ 0.95%, such as 0.91%, 0.92% or 0.95%.

[0022] In the present invention, in the tracer liposome solution, the content of the tracer can be routinely adjusted on the premise of meeting the visual recognition of the staining effect. The content of the tracer can be ≥ 2 mg / mL, preferably ≥ 4 mg / mL, more preferably ≥ 9 mg / mL, such as 10.1 mg / mL or 10.3 mg / mL. The content of the tracer generally refers to the content (mg) of the tracer in the tracer liposome solution (1 mL).

[0023] In the present invention, in the tracer liposome solution, the liposome encapsulation efficiency can be ≥ 98.9%, such as 99.2%, 99.3%, 99.4%, 99.5% or 99.6%.

[0024] The present invention provides a method for preparing a tracer liposome solution, which comprises the following steps:

[0025] S1 Inject the phospholipid solution into the inner aqueous phase solution, age, extrude, and ultrafilter to obtain a blank liposome solution;

[0026] Wherein, the phospholipid solution includes phospholipids, cholesterol and a solvent; the phospholipids include one or more of distearoyl phosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC) and sphingomyelin (SM);

[0027] S2 Add the blank liposome solution to the tracer solution for mixing and encapsulation, and ultrafilter;

[0028] Wherein, the encapsulation time is not less than 5 min, and during encapsulation, the pH value of the system is 6 - 11.

[0029] In the present invention, the tracer liposome solution is preferably as described above.

[0030] In S1, the phospholipid solution can be prepared by conventional methods in the art. For example, phospholipids and cholesterol are dissolved in a solvent and heated with stirring for dissolution. The heating temperature can be not lower than 60 °C, such as 63 °C, 68 °C or 73 °C.

[0031] In S1, in the phospholipid solution, the phospholipid is preferably distearoyl phosphatidylcholine and / or hydrogenated soy phospholipid.

[0032] In S1, in the phospholipid solution, the concentration of the phospholipid can be 237-711 mg / mL, preferably 400-800 mg / mL, such as 423 mg / mL, 474 mg / mL, 500 mg / mL, 708 mg / mL or 750 mg / mL.

[0033] In S1, in the phospholipid solution, the mass ratio of phospholipid to cholesterol can be (1-10):1, preferably (2-9):1, such as 3:1, 4:1, 5:1 or 7:1. Generally, the mass ratio of phospholipid to cholesterol in the phospholipid solution is the same as that in the tracer liposome.

[0034] In S1, in the phospholipid solution, the solvent can be a solvent that can conventionally dissolve phospholipids and cholesterol in the art, such as absolute ethanol.

[0035] In S1, the volume ratio of the phospholipid solution to the inner aqueous phase solution can be 1:(2-20), preferably 1:5, 1:10 or 1:15.

[0036] In S1, the inner aqueous phase solution can be prepared by conventional methods in the art. For example, the encapsulant is dissolved in water and heated with stirring for dissolution. The heating temperature can be not lower than 60 °C, such as 63 °C, 68 °C or 73 °C.

[0037] In S1, in the inner aqueous phase solution, the type of the encapsulant can be ammonium sulfate and / or sucrose octasulfate, preferably ammonium sulfate.

[0038] In S1, in the inner aqueous phase solution, the concentration of the encapsulant can be 0.2-1 M, preferably 0.2-0.6 M, such as 0.4 M or 0.5 M.

[0039] In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the temperatures of both solutions are generally not lower than 60 °C, such as 63 °C, 68 °C or 73 °C.

[0040] In S1, those skilled in the art can routinely adjust the injection time according to the volumes of the phospholipid solution and the internal aqueous phase solution. In a preferred embodiment, when injecting the phospholipid solution into the internal aqueous phase solution, the injection time is 5 min.

[0041] In S1, when injecting the phospholipid solution into the internal aqueous phase solution, the injection time can be 5 - 10 min.

[0042] In S1, when injecting the phospholipid solution into the internal aqueous phase solution, the injection pore size can be conventional in the art, such as 0.6 mm. The injection pore size generally refers to the fluid channel size when the phospholipid solution is injected into the internal aqueous phase through a syringe needle or a microfluidic chip channel.

[0043] In S1, when injecting the phospholipid solution into the internal aqueous phase solution, the rotation speed of stirring the internal aqueous phase solution can be conventional in the art, such as 300 rpm.

[0044] In S1, the aging time is at least 5 min, can be 5 min - 2 h, preferably 15 - 60 min, such as 30 min.

[0045] In S1, the aging temperature can be 63°C - 73°C, such as 63°C, 68°C or 73°C.

[0046] In S1, during the aging process, the rotation speed can be 150 - 250 rpm, such as 200 rpm.

[0047] In S1, the extrusion operation and conditions can be conventional in the art. Preferably, it is extruded through a polycarbonate membrane with a pore size of 200 nm, and then through a polycarbonate membrane with a pore size of 80 nm. During the extrusion process, the number of membrane layers and the number of extrusion times can be routinely adjusted according to the size of the finished product particle size.

[0048] In a preferred embodiment, first use a 200 nm * 3 (number of layers) polycarbonate membrane to extrude once, and then use an 80 nm * 3 (number of layers) polycarbonate membrane to extrude 3 times. The average particle size of the obtained finished product is 70 nm.

[0049] In a preferred embodiment, use an 80 nm * 3 (number of layers) polycarbonate membrane to extrude 2 times until the average particle size is 80 nm.

[0050] In a preferred embodiment, first use a 200 nm * 3 (number of layers) polycarbonate membrane to extrude once, and then use an 80 nm * 3 (number of layers) polycarbonate membrane to extrude once. The average particle size range of the obtained finished product is 131 nm.

[0051] In a preferred embodiment, first use a 200 nm * 3 (number of layers) polycarbonate membrane to extrude once, and then use an 80 nm * 1 (number of layers) polycarbonate membrane to extrude once. The average particle size of the obtained finished product is 180 nm.

[0052] In a preferred embodiment, a 200 nm × 3 (number of layers) polycarbonate membrane is used for extrusion once, and the average particle size of the obtained product is 200 nm.

[0053] In S1, after the extrusion and before the ultrafiltration, the temperature of the system can be lowered to ≤25°C.

[0054] In S1, the operations and conditions of the ultrafiltration can be conventional in the art. Generally, normal temperature injection water is used as the external aqueous phase for ultrafiltration. The purpose of the ultrafiltration is generally to remove the unencapsulated encapsulant and provide a concentration difference for the subsequent encapsulation operation.

[0055] In S1, the ultrafiltration can be carried out by constant volume concentration ultrafiltration using a 300 KD ultrafiltration membrane.

[0056] In S1, the number of ultrafiltration times can be conventional in the art, for example, ≥8 times.

[0057] In S2, in the blank liposome solution, the actually detected concentration of the encapsulant can be 0.04 - 0.09 M, such as 0.05 M, 0.0625 M or 0.0833 M. The actually detected concentration of the encapsulant refers to the concentration of the encapsulant remaining in the blank liposome solution after removing the unencapsulated encapsulant by ultrafiltration in step S1, that is, the actual content of the encapsulant in the blank liposome solution when mixed with the tracer solution.

[0058] In S2, the tracer solution can be prepared by conventional methods in the art. For example, the tracer and the solvent are mixed and heated to dissolve. The temperature for heating and dissolving can be 35 - 45°C, such as 40°C.

[0059] In S2, when the blank liposome solution is added to the tracer solution, the temperature of the tracer solution can be 30 - 40°C, such as 35°C.

[0060] In S2, in the tracer solution, the tracer can be one or more of methylene blue, indocyanine green, and mitoxantrone, such as methylene blue, indocyanine green, or mitoxantrone, preferably methylene blue.

[0061] In S2, in the tracer solution, the solvent can be injection water.

[0062] In S2, in the tracer solution, the mass percentage concentration of the tracer does not exceed 3%, such as 1.48%.

[0063] In S2, the molar ratio of the tracer in the tracer solution to the actually detected encapsulant in the blank liposome solution can be ≥ (1:1), preferably ≥ (1.5:1), such as 2.0:1, 2.5:1, or 3:1. The actually detected encapsulant refers to the encapsulant remaining in the blank liposome solution after ultrafiltration to remove the unencapsulated encapsulant in step S1. When the above molar ratio ≤ 2:1, the above molar ratio is generally the same as the molar ratio of the tracer to the encapsulant in the tracer liposome solution. When the above molar ratio > 2:1, the excessive tracer will be partially removed during the ultrafiltration in S2, and the molar ratio of the tracer to the encapsulant in the tracer liposome solution will generally be slightly greater than 2:1.

[0064] In S2, according to the actual content of the encapsulant detected in the blank liposome solution and the controlled molar ratio of the tracer to the encapsulant, those skilled in the art can routinely adjust the addition amount of the tracer solution.

[0065] In S2, those skilled in the art can routinely adjust the mixing time of the blank liposome solution and the tracer solution according to their volumes. In a preferred embodiment, the blank liposome solution is added to the tracer solution and mixed for 5 min, and then the pH value of the system is adjusted for encapsulation. Generally, the pH value of the system is adjusted with hydrochloric acid or sodium hydroxide solution (the concentration can be 2M).

[0066] In S2, after adjusting the pH value of the mixture of the blank liposome solution and the tracer solution to reach the encapsulation pH value and the temperature to reach the encapsulation temperature, encapsulation is started.

[0067] In S2, the temperature of the encapsulation can be not lower than 55°C, preferably 60 - 76°C, more preferably 65 - 70°C, such as 68°C.

[0068] In S2, the encapsulation time is preferably 5 min - 60 min, such as 10 - 55 min, such as 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, or 50 min.

[0069] In S2, during the encapsulation, the pH value of the system is preferably 6.5 - 10.5, such as 7, 7.5, 8, 8.5, 9, 9.8, 10, 10.5, or 11.

[0070] In S2, after the encapsulation, it is generally cooled to ≤ 25°C.

[0071] In S2, the operations and conditions of the ultrafiltration can be conventional in the art. In the present invention, the purpose of the ultrafiltration is generally to remove the incompletely encapsulated drug and provide an external environment with the same osmotic pressure and pH as that of the human body.

[0072] In S2, the ultrafiltrate of the ultrafiltration can be an aqueous solution of a mixture of glucose and HEPES, an aqueous solution of a mixture of sodium dihydrogen phosphate and glucose, or an aqueous solution of a mixture of sodium chloride and HEPES.

[0073] In the aqueous solution of the mixture of glucose and HEPES, the concentration of HEPES can be 6 - 8 mg / mL, such as 4.05 mg / mL. The concentration of glucose can be 40 - 50 mg / mL, such as 47 mg / mL.

[0074] In the aqueous solution of the mixture of sodium dihydrogen phosphate and glucose, the concentration of sodium dihydrogen phosphate can be 6 - 8 mg / mL, such as 6.8 mg / mL. The concentration of glucose can be 40 - 50 mg / mL, such as 47 mg / mL.

[0075] In the aqueous solution of the mixture of sodium chloride and HEPES, the concentration of sodium chloride can be 5 - 12 mg / ml, such as 8.45 mg / mL. The concentration of HEPES can be 3 - 5 mg / ml, such as 4.1 mg / mL.

[0076] In S2, the pH value of the ultrafiltrate of the ultrafiltration can be 2 - 11, preferably 3 - 10, such as 4, 6.5, 7, 8 or 9.

[0077] In S2, the ultrafiltration can be carried out by constant volume concentration ultrafiltration using a 300KD ultrafiltration membrane.

[0078] In S2, the number of times of the ultrafiltration can be ≥10 times.

[0079] The present invention also provides a tracer liposome solution prepared by the preparation method as described above.

[0080] In the present invention, in the tracer liposome solution, the particle size of the liposome can be not less than 70 nm, preferably 70 - 200 nm, more preferably 80 - 200 nm, such as 90 nm, 100 nm, 131 nm, 150 nm or 180 nm.

[0081] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0082] The reagents and raw materials used in the present invention are all commercially available.

[0083] The positive and progressive effects of the present invention are as follows:

[0084] The tracer liposome solution of the present invention not only has high recognition during clinical use, small local irritation during clinical use, and relatively fast local dissipation after use, but also can have a relatively high drug loading capacity.

[0085] In some preferred embodiments, the tracer liposome of the present invention not only has a relatively high drug loading capacity, but also can have a relatively high encapsulation efficiency and / or stability. Description of the Drawings

[0086] Figure 1 It is a diagram showing the tracing of rat lymph nodes with liposome solutions of different final product particle sizes. Among them, Figure 1 Part A of - Figure 1 Part C of have final product particle sizes of 70 nm (Example 59), 200 nm (Example 60), and 131 nm (Example 1), respectively.

[0087] Figure 2 It is a diagram showing the tracing of rats with different encapsulants. Figure 2 Part A of uses ammonium sulfate as the inner aqueous phase encapsulation in Example 1, Figure 2 Part B of uses sucrose octasulfate as the inner aqueous phase encapsulation in Example 62.

[0088] Figures 3 - 4 It is a comparison diagram of the staining of rat lymph nodes after encapsulation with different dyes using the same process. Among them, Figure 3 Part A of is the mitoxantrone liposome solution prepared in Example 63, Figure 3 Part B of, Figure 4 Part B of are both methylene blue liposome solutions prepared in Example 1, Figure 4 Part A of is the indocyanine green liposome solution prepared in Example 64.

[0089] Figure 5 It is a comparison diagram of the tracing of rat lymph nodes between a commercially available methylene blue injection and the methylene blue liposome solution of Example 1.

[0090] Figure 6 It is a comparison diagram of the subcutaneous irritation of a commercially available methylene blue injection, a mitoxantrone hydrochloride injection, and the methylene blue liposome solution of Example 1 in rats.

[0091] Figure 7 It is a comparison diagram of the irritation difference between the methylene blue liposome solution of Example 1 and a commercially available nano-carbon suspension injection.

[0092] Figure 8 It is a photo of the free drug of the product passing through a gel column after encapsulation with different molar ratios of methylene blue and ammonium sulfate (without performing step S2 ultrafiltration) (during the gel column passing in the intermediate encapsulation efficiency test). Among them, Figure 8 Part A of - Figure 8 In parts C of, the molar ratios of methylene blue to ammonium sulfate are 2.5:1 (Example 17), 2.0:1 (Example 1), and 1.5:1 (Example 16), respectively. Detailed Description of the Invention

[0093] The present invention will be further illustrated by way of examples below, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0094] In the following examples or comparative examples, the nano-carbon suspension injection (trade name: Canaline) was purchased from Chongqing Lummy Pharmaceutical Co., Ltd.; mitoxantrone hydrochloride (trade name: Futashu) was purchased from Shenzhen China Resources Nine Creation Pharmaceutical Co., Ltd.; methylene blue injection was purchased from Jichuan Pharmaceutical Group Co., Ltd.

[0095] In the present invention:

[0096] The content of the degraded impurities of the finished phospholipid (content of finished LPC) generally refers to the content (mg) of the degraded impurities of the phospholipid in the liposome solution (1 mL).

[0097] The content of the degraded impurities of the phospholipid in the blank liposome solution (content of LPC in the blank liposome solution) is the content (mg) of LPC in the blank liposome solution (1 mL) obtained after ultrafiltration in step S1. The specific test method is the same as that for detecting the content of finished LPC.

[0098] The content of finished LPC at day 0 generally refers to the content of LPC of the liposome solution prepared in the experiment without any treatment and directly tested.

[0099] The content of LPC under the influence factor at 40 °C generally refers to the content of LPC tested after the finished liposome solution is sealed and stored at 40 °C and an environmental humidity of 75% RH (75% humidity) for 14 days.

[0100] The content of the tracer generally refers to the content (mg) of the tracer in the liposome solution (1 mL).

[0101] The encapsulation efficiency at day 0 generally refers to the encapsulation efficiency calculated by directly testing the liposome solution prepared in the experiment without any treatment.

[0102] The encapsulation efficiency during the encapsulation process refers to the intermediate encapsulation efficiency, that is, the encapsulation efficiency of the liposome solution before ultrafiltration in step S2.

[0103] The particle size of the finished product at day 0 generally refers to the particle size directly detected without any treatment of the liposome solution prepared in the experiment.

[0104] Test method for physical stability in 0 days: Take 0.5 mL of liposome solution (the liposome solution prepared in the experiment was directly detected without any treatment), centrifuge (10000 rpm for 5 min) and observe. If no obvious precipitation can be seen with the naked eye, it indicates physical stability; if solid precipitation is seen at the bottom with the naked eye, the solid precipitate is the sediment. If there is a very small amount of sediment, it indicates relatively physical stability. If there is more sediment, it indicates physical instability.

[0105] "Encapsulation efficiency under 40 °C influencing factors" refers to the encapsulation efficiency measured after storing for 14 days at 40 °C and an environmental humidity of 75% RH (75% humidity) after the liposome solution is packaged in a conventional vial and sealed with a sealing cap.

[0106] The drug-lipid ratio generally refers to the ratio of the tracer concentration (mg / ml) to the phospholipid concentration (mg / ml) in the finished tracer liposome solution. The tracer concentration can be detected by the detection method of methylene blue content in item "2" of Example 1, and the phospholipid concentration can be detected by the detection method of phospholipid degradation impurity content in item "1" of Example 1.

[0107] Example 1 Preparation of methylene blue liposome solution

[0108] S1 Inject the phospholipid solution into the inner aqueous phase solution, age, extrude, and perform one-time ultrafiltration to obtain a blank liposome solution; where:

[0109] Preparation of phospholipid solution: Take 63.14 g of DSPC and 21.05 g of cholesterol and dissolve them in 133 ml of absolute ethanol, and keep in a water bath at 68 °C ± 5 °C. In the phospholipid solution, the phospholipid concentration is 474 mg / mL. The mass ratio of phospholipid to cholesterol is 3:1.

[0110] Preparation of inner aqueous phase solution: Take 88.01 g of ammonium sulfate and dissolve it in 1333 g of injection water, and heat up to 68 °C. The volume ratio of the phospholipid solution to the inner aqueous phase solution is 1:10, and the concentration of the ammonium sulfate solution is 0.5 M.

[0111] Emulsification injection: Keep the solution temperature at 68 °C ± 2 °C, slowly inject the phospholipid solution into the inner aqueous phase solution, the injection pore diameter is 0.6 mm, the stirring speed of the inner aqueous phase is 300 rpm, and the injection time is about 5 min.

[0112] Aging: After injection, keep the emulsion at 68 °C ± 5 °C for 30 min, and the rotation speed is 200 rpm.

[0113] Extrusion: First, extrude once with a 200 nm * 3 polycarbonate membrane, and then extrude once with an 80 nm * 3 polycarbonate membrane. The average particle size range of the obtained product is 100 - 150 nm. After extrusion, the sample is cooled to ≤ 25 °C and waiting for ultrafiltration.

[0114] Primary ultrafiltration: Using normal-temperature injection water as the external aqueous phase, a 300KD ultrafiltration membrane is used for constant-volume concentration ultrafiltration, and the number of ultrafiltration times is 8 times.

[0115] S2 After adding the blank liposome solution to the API solution and mixing for 5 min, adjust the pH to 9.8 with 2M sodium hydroxide solution, quickly heat up to 68 °C, and maintain for 15 min (start the encapsulation process immediately after adjusting the pH, that is, the encapsulation pH is 9.8, the encapsulation temperature is 68 °C, and the encapsulation time is 15 min), then quickly cool down to ≤25 °C and perform secondary ultrafiltration to obtain methylene blue liposome solution. Among them:

[0116] Preparation of API solution: Take 40 g of methylene blue (calculated as anhydrous), add 2660 g of injection water, heat to 40 °C to dissolve, and after dissolution, cool down to maintain the solution temperature at 35 °C. In the API solution, the mass percentage concentration of methylene blue is 1.48%.

[0117] According to the actual content of ammonium sulfate detected in the blank liposome solution and the controlled molar ratio of methylene blue to ammonium sulfate, adjust the addition amount of the API solution. In Example 1, during the mixing and encapsulation process, the actual concentration of ammonium sulfate in the blank liposome solution is 0.0625 M, and the set molar ratio of methylene blue to ammonium sulfate is 2:1, and the volume of the methylene blue solution can be further calculated.

[0118] ‌Preparation of secondary ultrafiltrate: The ultrafiltrate is an aqueous solution of glucose and HEPES. Take 4.7 g of glucose and 0.405 g of HEPES, add water and stir to dissolve, then dilute to 100 ml, and adjust the pH to 6.5 with sodium hydroxide.

[0119] Secondary ultrafiltration: Using normal-temperature secondary ultrafiltrate as the external aqueous phase, a 300KD ultrafiltration membrane is used for constant-volume concentration ultrafiltration, and the number of ultrafiltration times is 10 times.

[0120] Stability test of methylene blue liposome solution in Example 1

[0121] According to the method of Example 1, repeat the experiment three times to obtain methylene blue liposome solutions of batches 1-3 respectively. The above three batches of methylene blue liposome solutions are tested according to the following methods respectively.

[0122] 1. Under the accelerated conditions of 25 °C and 75% RH (75% humidity), at each time point, prepare the test solution of the sample according to the following method and detect the phospholipid degradation impurity (LPC): Take a precise volume of 1 ml of the liposome solution in a 5 ml volumetric flask, dilute to the scale with a diluent (methanol), shake well, centrifuge at 10000 rpm for 10 min, and inject the sample for detection according to the following table.

[0123] Table 1 Test conditions

[0124]

[0125] The test results are shown in the following table.

[0126] Table 2 LPC content (mg / ml) of the finished product in the accelerated test at 25°C and 75% RH

[0127]

[0128] 2. Under the accelerated conditions of 25°C and 75% RH (75% humidity), the test samples at each time point were prepared into test solutions and the methylene blue content and impurity content were detected according to the following methods.

[0129] Precisely measure 1 ml of liposome solution into a 100-ml volumetric flask, first add 9 ml of methanol to break the emulsion, and finally dilute to the mark with a diluent (25% acetonitrile solution). Shake well, centrifuge at 10,000 rpm for 10 min, and inject samples for detection according to the following table.

[0130] Table 3 Test conditions

[0131]

[0132] The test results are shown in the following table.

[0133] Table 4 Total impurities of related substances in the accelerated test at 25°C and 75% RH

[0134]

[0135] Table 5 Methylene blue content in the accelerated test at 25°C and 75% RH

[0136]

[0137] 3. Under the accelerated conditions of 25°C and 75% RH (75% humidity), the encapsulation efficiency of the test samples at each time point was calculated according to the following methods.

[0138] According to the test solution and detection method in the foregoing "2. Detection of methylene blue content and impurity content", take 100 μL of the test sample and pass it through a gel column. After separating the free and liposome forms, detect the free and liposome concentrations according to the content detection method. Then, encapsulation efficiency = liposome content / (free content + liposome content). The calculation results are shown in the following table.

[0139] Table 6 Encapsulation efficiency in the accelerated test at 25°C and 75% RH

[0140]

[0141] 4. Under the accelerated conditions of 25°C and 75% RH (75% humidity), the particle size of the test samples at each time point was detected according to the following methods.

[0142] Take 10 μL of the test solution in the aforementioned "2. Detection of methylene blue content and impurity content", add it to 10 mL of pure water, mix well, and take a sample to detect the particle size with a dynamic light scattering particle size detector. The particle size results are shown in Table 7.

[0143] Table 7 Particle size in the accelerated test at 25°C and 75% RH

[0144]

[0145] Unless otherwise specified, the test methods for the LPC content, total impurities of related substances, methylene blue content, encapsulation efficiency, and particle size of the liposome solution in the present invention are the same as those in Example 1.

[0146] Examples 2 - 3, Comparative Examples 1 - 2 Phospholipid types

[0147] In Examples 2 - 3 and Comparative Examples 1 - 2, the phospholipid types are as shown in the following table. Other operations and conditions are the same as those in Example 1. Liposome solutions are prepared respectively, and the test results are shown in the following table.

[0148] Table 8 Screening of phospholipid types

[0149]

[0150] In the table, the test data are all the test data of the final finished liposome solution. "NA" means that after the system settles, this data cannot be tested.

[0151] It can be seen that in the above examples, the drug - lipid ratio of the liposome can reach 69%. When the phospholipid is DSPC or HSPC, the encapsulation efficiency is higher, the stability is better, and the drug - lipid ratio is higher.

[0152] Examples 4 - 5 Mass ratio of phospholipid to cholesterol

[0153] In Examples 4 - 5, the mass ratios of phospholipid to cholesterol are 1:1 and 10:1 respectively. Other operations and conditions are the same as those in Example 1. Liposome solutions are prepared respectively, and the test results are shown in the following table.

[0154] Table 9 Investigation of the mass ratio of phospholipid to cholesterol

[0155]

[0156] In Table 9, except for the encapsulation efficiency during the encapsulation process, other test data are all the test data of the final finished liposome solution.

[0157] It can be seen that in the above examples, the drug - lipid ratio of the liposome can reach more than 78%. When the mass ratio of phospholipid to cholesterol is 3 - 5:1, the encapsulation efficiency is higher, the stability is better, and the drug - lipid ratio is higher.

[0158] Examples 6 - 7 Volume Ratio of Phospholipid Solution to Inner Aqueous Phase Solution

[0159] In Examples 6 - 7, the volume ratios of the phospholipid solution to the inner aqueous phase solution were 1:2 and 1:20 respectively. Other operations and conditions were the same as those in Example 1, and liposome solutions were prepared respectively. The test results are shown in the following table.

[0160] Table 10 Investigation of Volume Ratio of Phospholipid Solution to Inner Aqueous Phase Solution

[0161]

[0162] In the table, all test data are the test data of the final finished liposome solution.

[0163] It can be seen that in the above examples, the drug - lipid ratio of the liposome can reach more than 77%. When the volume ratio of the phospholipid solution to the inner aqueous phase solution is 1:(2 - 20), the encapsulation efficiency and stability of the obtained product meet the requirements.

[0164] Examples 8 - 9 Phospholipid Concentration

[0165] In Examples 8 - 9, the amounts of absolute ethanol in the phospholipid solution were 266 mL and 89 mL respectively, and the concentrations of phospholipids in the phospholipid solution were 237 mg / ml and 708 mg / ml respectively. Other operations and conditions were the same as those in Example 1, and liposome solutions were prepared respectively. The test results are shown in the following table.

[0166] Table 11 Investigation of Phospholipid Concentration

[0167]

[0168] Among them, all test data are the test data of the final finished liposome solution.

[0169] It can be seen that in the above examples, the drug - lipid ratio of the liposome can reach 79%. When the concentration of the phospholipid solution is in the range of 200 - 800 mg / mL, the liposome solution has better encapsulation efficiency and stability.

[0170] Examples 10 - 11 Concentration of Encapsulating Agent

[0171] In Examples 10 - 11, the preparation and concentration of the ammonium sulfate solution are shown in the following table. Other operations and conditions were the same as those in Example 1, and liposome solutions were prepared respectively. The test results are shown in the following table.

[0172] Table 12 Investigation of Concentration of Encapsulating Agent

[0173]

[0174] In the table, the test data are all the test data of the final finished liposome solution.

[0175] As can be seen, in the above embodiments, the drug-lipid ratio of the liposome can reach more than 53%. When the ammonium sulfate solution is in the range of 0.2 - 1 M, the liposome solution has relatively excellent encapsulation efficiency, stability, and drug-lipid ratio. When the ammonium sulfate solution is 0.5 - 1 M, the drug-lipid ratio effect is optimal.

[0176] Examples 12 - 1, 12 - 2, 13 - 15 Aging time

[0177] In Examples 12 - 1, 12 - 2, and 13 - 15, the aging times are shown in the following table respectively. Other operations and conditions are the same as those in Example 1, and liposome solutions are prepared respectively. The test results are shown in the following table.

[0178] Table 13 Investigation of aging time

[0179]

[0180] As can be seen, in the above embodiments, the drug-lipid ratio of the liposome can reach more than 69%. When the aging time is 15 - 60 min, the blank liposome solution is more stable, the physical stability of the finished liposome solution is good, and the drug-lipid ratio content is relatively high.

[0181] Examples 16 - 17 Methylene blue and ammonium sulfate molar ratio

[0182] In Examples 16 - 17, during the encapsulation process, the actual concentration of ammonium sulfate in the blank liposome solution, and the molar ratio of methylene blue to ammonium sulfate are shown in the following table respectively. Other operations and conditions are the same as those in Example 1, and liposome solutions are prepared respectively. The test results are shown in the following table.

[0183] Table 14 Investigation of methylene blue and ammonium sulfate molar ratio

[0184]

[0185] As can be seen, in the above embodiments, the drug-lipid ratio of the liposome can reach more than 59%. When the molar ratio of methylene blue to ammonium sulfate is 2.0 - 2.5:1, the LPC content in the liposome solution is lower, the stability is better, and the drug-lipid ratio is higher.

[0186] Examples 18 - 53 Influence of encapsulation time, temperature, and pH

[0187] In Examples 18 - 53, the encapsulation time, temperature, and pH value are shown in the following table respectively. Other operations and conditions are the same as those in Example 1, and liposome solutions are prepared respectively. The test results are shown in the following table.

[0188] Table 15 Influence of encapsulation time, temperature, and pH

[0189]

[0190] In the table, the above data are all the test results of the finished liposome solution.

[0191] It can be seen that the drug-lipid ratio in the liposome solution of the present invention is all greater than 42%. On the premise of the same encapsulation temperature and encapsulation time, when the pH value is between 9 and 11, the drug-lipid ratio in the liposome solution is higher.

[0192] Example 54 - 58 External aqueous phase pH value

[0193] In Examples 54 - 58, the pH value of the external aqueous phase was adjusted as shown in the following table respectively. Other operations and conditions were the same as those in Example 1. Liposome solutions were prepared respectively, and the test results are shown in the following table.

[0194] Table 16 Investigation of the pH value of the external aqueous phase

[0195]

[0196] In the table, the above data are all the test results of the finished liposome solution.

[0197] It can be known from this that in the above examples, the drug-lipid ratio of the liposome can reach more than 79%. When the pH value of the external aqueous phase is between 4 and 9, the liposome solution is more stable.

[0198] Example 59

[0199] The difference from Example 1 is only in the extrusion process: first extrude once with a 200 nm * 3 polycarbonate membrane, and then extrude three times with an 80 nm * 3 polycarbonate membrane. The average particle size of the obtained product is 70 nm. Other operations and conditions are the same as those in Example 1, and a liposome solution is prepared.

[0200] Example 60

[0201] The difference from Example 1 is only in the extrusion process: extrude once with a 200 nm * 3 polycarbonate membrane. The average particle size of the obtained product is 200 nm. Other operations and conditions are the same as those in Example 1, and a liposome solution is prepared.

[0202] Example 61

[0203] The difference from Example 1 is only in the extrusion process: extrude twice with an 80 nm * 3 polycarbonate membrane. The average particle size of the obtained product is 80 nm. Other operations and conditions are the same as those in Example 1, and a liposome solution is prepared.

[0204] Example 62

[0205] It is only different from Example 1 in that: first, extrude once with a 200 nm * 3 polycarbonate membrane, and then extrude once with an 80 nm * 1 polycarbonate membrane. The average particle size of the obtained finished product is 180 nm. Other operations and conditions are the same as those in Example 1, and a liposome solution is prepared.

[0206] Example 63

[0207] Replace ammonium sulfate in Example 1 with sucrose octasulfate in equal amount, and keep other operations and conditions unchanged to prepare a liposome solution.

[0208] Example 64

[0209] Replace methylene blue in Example 1 with mitoxantrone in equal amount, and keep other operations and conditions the same as those in Example 1 to prepare a mitoxantrone liposome solution.

[0210] Example 65

[0211] Replace methylene blue in Example 1 with indocyanine green in equal amount, and keep other operations and conditions the same as those in Example 1 to prepare an indocyanine green liposome solution.

[0212] The drug-lipid ratio and the LPC content of the finished product on day 0 of different tracer liposome solutions are shown in Table 17 below.

[0213] Table 17 Effects of different tracer liposomes

[0214]

[0215] It can be seen that in terms of the drug-lipid ratio and the LPC content of the finished product on day 0, the methylene blue liposome solution in Example 1 is the best, followed by the indocyanine green liposome in Example 65 and the mitoxantrone liposome in Example 64.

[0216] Example 66

[0217] Replace the secondary ultrafiltrate in Example 1 with a mixed aqueous solution of sodium chloride and HEPES, and keep other operations and conditions the same as those in Example 1.

[0218] The preparation method of the mixed aqueous solution is as follows: take 421 g of sodium chloride and 202.5 g of HEPES, add 49.4 kg of injection water, stir to dissolve, adjust the pH value to 6.5 with sodium hydroxide, and stir for 5 minutes for later use. In the ultrafiltrate, the concentration of sodium chloride is 8.45 mg / mL, and the concentration of HEPES is 4.1 mg / mL.

[0219] Example 67

[0220] Replace the secondary ultrafiltrate in Example 1 with a mixed aqueous solution of sodium dihydrogen phosphate and glucose, and keep other operations and conditions the same as those in Example 1.

[0221] The method for preparing the mixed aqueous solution is as follows: 0.68 g of sodium dihydrogen phosphate, 0.06 g of sodium hydroxide, and 4.7 g of glucose are diluted with water to 100 ml. The pH value of the system is 6.5.

[0222] The drug-lipid ratio and the LPC content of the influencing factor at 40 °C of the tracer liposome solution prepared from the ultrafiltrate of different secondary ultrafiltrations are shown in the following table.

[0223] Table 18 Effects of different tracer liposomes

[0224]

[0225] In the tracer liposome solution prepared in the examples of the present invention, the content of the tracer is about 10 mg / mL.

[0226] Figures 1 - 5 The process of the animal experiment: After weighing the SD rats, they were anesthetized by intraperitoneal injection of 5% chloral hydrate (the injection dose was according to 0.7 mL / 100 g). The skin of the root of the hind limb thigh of the rats was depilated, and the drug was injected subcutaneously at the sole of the hind limb. 20 μL of the tracer was injected (completed within 50 - 60 s), and the sole was pressed for 3 min after injection. After 30 min of drug administration, the skin of the thigh was cut open, and the stained popliteal lymph ( Figures 1 - 4 ) was found in the muscle gap of the rat leg. In the same way, it was injected into the forelimb to find the axillary lymph node ( Figure 5 ).

[0227] Figure 1 It is the tracer diagram of the rat lymph nodes of the liposome solution with different finished product particle sizes. Among them, Figure 1 Part A of - Figure 1 Part C of the finished product particle sizes are 70 nm (Example 59), 200 nm (Example 60), and 131 nm (Example 1) respectively. As can be seen from Figure 1 , when the particle size is 70 nm, the smudging is serious. When the particle size is 131 nm and 200 nm, the sentinel lymph node staining is full.

[0228] During the actual use of the liposome solution obtained in Examples 61 - 62, it is similar to that in Example 1 and Example 60, without smudging and with full sentinel lymph node staining.

[0229] Figure 2 It is the tracer picture of different encapsulants for rats. Figure 2 Part A of is that ammonium sulfate is used as the inner aqueous phase encapsulation in Example 1, Figure 2 Part B of is that sucrose octasulfate is used as the inner aqueous phase encapsulation in Example 62. As can be seen from Figure 2 , the staining of the liposome encapsulated with ammonium sulfate is clearer.

[0230] Figures 3 - 4Comparison diagram of the staining of rat lymph nodes after encapsulation of different dyes using the same process package. Among them, Figure 3 Part A of Figure 3 is the mitoxantrone liposome solution prepared in Example 63, Figure 3 Part B of Figure 3 , Figure 4 Part B of Figure 4 are both methylene blue liposome solutions prepared in Example 1, Figure 4 Part A of Figure 4 is the indocyanine green liposome solution prepared in Example 64. As can be seen from the figure, using the preparation method of the present invention, methylene blue, mitoxantrone, and indocyanine green can all achieve encapsulation and in vivo lymph node tracing.

[0231] Figure 5 Comparison diagram of the lymph node tracing of commercially available methylene blue injection and methylene blue liposome solution in Example 1 in rats. As can be seen from the figure, the secondary lymph nodes were not stained by the methylene blue liposome solution in Example 1.

[0232] Figure 6 Animal experiment process: After weighing the SD rats, they were anesthetized by intraperitoneal injection of 5% chloral hydrate (injection dose according to 0.7 mL / 100 g). The abdominal skin of the rats was depilated, and after subcutaneous injection of 20 μL of the tracer in the abdomen (injection completed in 50 - 60 s), observation was carried out 24 h later.

[0233] Figure 6 Comparison diagram of the subcutaneous irritation of commercially available methylene blue injection, mitoxantrone hydrochloride injection, and methylene blue liposome solution in Example 1 in rats. Figure 6 It shows that the local irritation of the methylene blue liposome solution in Example 1 is smaller.

[0234] Figure 7 Animal experiment process: After weighing the SD rats, they were anesthetized by intraperitoneal injection of 5% chloral hydrate (injection dose according to 0.7 mL / 100 g). After subcutaneous injection of 20 μL of the tracer in the plantar surface of the forelimb (injection completed in 50 - 60 s), observation was carried out 24 h later.

[0235] Figure 7 Comparison diagram of the irritation difference between the methylene blue liposome solution in Example 1 and commercially available nano-carbon suspension injection. Figure 7 It can be seen that the local irritation of the methylene blue liposome solution in Example 1 is smaller.

[0236] The above animal experiments show that the irritation of the liposome solution of the present invention used in lymph node tracing is much smaller than that of currently commercially available lymph node tracing varieties.

[0237] Figure 8 Photograph of the free drug of the product after encapsulation with different molar ratios of methylene blue and ammonium sulfate (without secondary ultrafiltration) passing through the gel column (during the intermediate encapsulation efficiency test, passing through the gel column). Among them, Figure 8 Part A of Figure 8 - Figure 8The molar ratios of methylene blue to ammonium sulfate in part C are 2.5:1 (Example 17), 2.0:1 (Example 1), and 1.5:1 (Example 16), respectively. From Figure 8 it can be seen that different molar ratios of methylene blue to sulfuric acid can all encapsulate methylene blue well, with less free drug.

Claims

1. A tracer liposome solution, characterized in that, It includes carrier liposomes and an aqueous tracer solution encapsulated within the carrier liposomes; Among them, the carrier liposomes include phospholipids, cholesterol, and an encapsulating agent; the phospholipids include one or more of distearoyl phosphatidylcholine, hydrogenated soy phospholipids, and sphingomyelin; the tracer is one or more of methylene blue, indocyanine green, and mitoxantrone.

2. The tracer liposome solution according to claim 1, wherein, The tracer liposome solution satisfies one or more of the following conditions: (1) The phospholipids include distearoyl phosphatidylcholine and / or hydrogenated soy phospholipids; (2) The mass ratio of the phospholipids to the cholesterol is (1 - 10):1, preferably (2 - 9):1, such as 3:1, 4:1, 5:1, or 7:1; (3) The encapsulating agent includes ammonium sulfate and / or sucrose octasulfate, preferably ammonium sulfate; (4) The tracer is methylene blue, indocyanine green, or mitoxantrone, preferably methylene blue; (5) The molar ratio of the tracer to the encapsulating agent ≥ (1:1), preferably ≥ (1.5:1), such as 2.0:1, 2.5:1, or 3:1; (6) In the tracer liposome solution, the particle size of the liposomes is not less than 70 nm, preferably 70 nm - 200 nm, more preferably 80 nm - 200 nm, such as 90 nm, 100 nm, 131 nm, 132 nm, 133 nm, 134 nm, 150 nm, or 180 nm.

3. The tracer liposome solution according to claim 1, characterized in that, The tracer liposome solution satisfies one or more of the following conditions: (1) The drug-lipid ratio ≥ 36%; the drug-lipid ratio refers to the ratio of the tracer concentration to the phospholipid concentration in the tracer liposome solution; (2) The LPC content < 1 mg / mL; (3) The total impurity content of related substances ≤ 0.95%; (4) The tracer content ≥ 2 mg / mL; (5) The liposome encapsulation efficiency ≥ 98.9%.

4. The tracer liposome solution according to claim 1, wherein, The tracer liposome solution satisfies one or more of the following conditions: (1) The drug-lipid ratio ≥ 42%, preferably 42% - 81%; such as 42%, 44%, 45%, 47%, 48%, 52%, 53%, 58%, 59%, 60%, 61%, 63%, 64%, 67%, 69%, 70%, 71%, 72%, 75%, 76%, 77%, 78%, 79%, 80%, or 81%; (2) The content of LPC < 0.76 mg / mL, such as 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.10 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.18 mg / mL, 0.21 mg / mL, 0.26 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.37 mg / mL, 0.40 mg / mL, 0.48 mg / mL, 0.5 mg / mL, 0.57 mg / mL or 0.76 mg / mL; (3) The total impurity content of related substances is 0.91%, 0.92% or 0.95%; (4) The content of the tracer ≥ 4 mg / mL, preferably ≥ 9 mg / mL, such as 10.1 mg / mL or 10.3 mg / mL; and, (5) The liposome encapsulation efficiency is 99.2%, 99.3%, 99.4%, 99.5% or 99.6%.

5. A method for preparing a tracer liposome solution, characterized in that, It includes the following steps: S1 Inject the phospholipid solution into the inner aqueous phase solution, age, extrude, and ultrafilter to obtain a blank liposome solution; Among them, the phospholipid solution includes phospholipids, cholesterol and a solvent; the phospholipids include one or more of distearoyl phosphatidylcholine, hydrogenated soy phospholipid and sphingomyelin; S2 Add the blank liposome solution to the tracer solution for mixing and encapsulation, and ultrafilter; Among them, the encapsulation time is not less than 5 min, and during encapsulation, the system pH value is 6 - 11.

6. The preparation method according to claim 5, characterized in that, The preparation method satisfies one or more of the following conditions: (1) In S2, the encapsulation temperature is not lower than 55 °C; (2) In S2, the encapsulation time is 5 min - 60 min; and, (3) In S2, during encapsulation, the system pH value is 6.5 - 10.

5.

7. The preparation method according to claim 5, characterized in that The preparation method satisfies one or more of the following conditions: (1) In S2, the encapsulation temperature is 60 - 76 °C, more preferably 65 - 70 °C, such as 68 °C; (2) In S2, the encapsulation time is 10 - 55 min, such as 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min or 50 min; and, (3) In S2, during encapsulation, the system pH value is 7, 7.5, 8, 8.5, 9, 9.8, 10, 10.5 or 11.

8. The preparation method according to claim 5, characterized in that, The preparation method satisfies one or more of the following conditions: In S1, the preparation method of the phospholipid solution includes: dissolving phospholipids and cholesterol in a solvent, heating, and stirring to dissolve; In S1, in the phospholipid solution, the phospholipids include distearoyl phosphatidylcholine and / or hydrogenated soy phospholipid; In S1, in the phospholipid solution, the concentration of phospholipid is 237 - 711 mg / mL, preferably 400 - 800 mg / mL, such as 423 mg / mL, 474 mg / mL, 500 mg / mL, 708 mg / mL or 750 mg / mL; In S1, in the phospholipid solution, the mass ratio of phospholipid to cholesterol is (1 - 10):1, preferably (2 - 9):1, such as 3:1, 4:1, 5:1 or 7:1; In S1, the volume ratio of the phospholipid solution to the inner aqueous phase solution is 1:(2 - 20), preferably 1:5, 1:10 or 1:15; In S1, the preparation method of the inner aqueous phase solution includes the following steps: dissolving the encapsulant in water, heating, and stirring to dissolve; In S1, in the inner aqueous phase solution, the types of encapsulant include ammonium sulfate and / or sucrose octasulfate, preferably ammonium sulfate; In S1, in the inner aqueous phase solution, the concentration of the encapsulant is 0.2 - 1 M, preferably 0.2 - 0.6 M, such as 0.4 M or 0.5 M; In S1, when injecting the phospholipid solution into the inner aqueous phase solution, the temperatures of both solutions are not lower than 60 °C, such as 63 °C, 68 °C or 73 °C; In S1, the ripening time is at least 5 min, preferably 5 min - 2 h, more preferably 15 - 60 min, such as 30 min; In S1, the ripening temperature is 63 °C - 73 °C, such as 63 °C, 68 °C or 73 °C; In S1, during the ripening process, the rotation speed is 150 - 250 rpm, such as 200 rpm; In S1, the extrusion operation includes: first extruding through a polycarbonate membrane with a pore size of 200 nm, and then extruding through a polycarbonate membrane with a pore size of 80 nm; In S1, ultrafiltration can be carried out by constant volume concentration ultrafiltration using a 300 KD ultrafiltration membrane; and, In S1, the number of ultrafiltration times ≥ 8 times.

9. The preparation method according to claim 5, wherein The preparation method satisfies one or more of the following conditions: (1) In S2, in the blank liposome solution, the actually detected concentration of the encapsulant is 0.04 - 0.09 M, such as 0.05 M, 0.0625 M or 0.0833 M; the actually detected concentration of the encapsulant refers to the concentration of the encapsulant remaining in the blank liposome solution after removing the unencapsulated encapsulant by ultrafiltration in step S1; (2) In S2, in the tracer solution, the tracer is one or more of methylene blue, indocyanine green and mitoxantrone, such as methylene blue, indocyanine green or mitoxantrone, preferably methylene blue; (3) In S2, in the tracer solution, the mass percentage concentration of the tracer does not exceed 3%, such as 1.48%; (4) In S2, the molar ratio of the tracer in the tracer solution to the actually detected encapsulant in the blank liposome solution ≥ (1:1), preferably ≥ (1.5:1), such as 2.0:1, 2.5:1 or 3:1; (5) In S2, the ultrafiltration ultrafiltrate is an aqueous solution of glucose and HEPES, an aqueous solution of sodium dihydrogen phosphate and glucose, or an aqueous solution of sodium chloride and HEPES; In the mixed aqueous solution of glucose and HEPES, the concentration of HEPES is preferably 3.5 - 5.5 mg / mL, such as 4.05 mg / mL; the concentration of glucose is preferably 40 - 50 mg / mL, such as 47 mg / mL; In the mixed aqueous solution of sodium dihydrogen phosphate and glucose, the concentration of sodium dihydrogen phosphate is preferably 6 - 8 mg / mL, such as 6.8 mg / mL; the concentration of glucose is preferably 40 - 50 mg / mL, such as 47 mg / mL; In the mixed aqueous solution of sodium chloride and HEPES, the concentration of sodium chloride is preferably 5 - 12 mg / ml, such as 8.45 mg / mL; the concentration of HEPES is preferably 3 - 5 mg / ml, such as 4.1 mg / mL; (9) In S2, the pH value of the ultrafiltrate of the ultrafiltration is 2 - 11, preferably 3 - 10, such as 4, 6.5, 7, 8 or 9; (6) In S2, the ultrafiltration is carried out by constant volume concentration ultrafiltration using a 300KD ultrafiltration membrane; and, (7) In S2, the number of times of the ultrafiltration is ≥10 times.

10. A tracer liposome solution prepared by the preparation method according to any one of claims 5 - 9.

Images