Tracer liposome solution and preparation method therefor

Abstract

A tracer liposome solution and a preparation method therefor. The tracer liposome solution comprises a carrier liposome and an aqueous solution of a tracer encapsulated within 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 is one or more of methylene blue, indocyanine green, and mitoxantrone. The tracer liposome solution exhibits high identifiability, low local irritation, and relatively rapid local dissipation in clinical use, and may have a high drug loading capacity, or high encapsulation efficiency and / or stability.

Description

A tracer liposome solution and its preparation method

[0001] This application claims priority to Chinese patent application 2025104618711, filed on April 14, 2025, and Chinese patent application 202510461875X, filed on April 14, 2025. The full text of both of these Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to a tracer liposome solution and its preparation method. Background Technology

[0003] The methylene blue preparation used clinically is the methylene blue injection solution. During clinical use, it can cause significant local irritation and pain for patients. Furthermore, the methylene blue takes a long time to dissipate at the injection site, affecting patients' daily lives. Currently used tracers include nano-carbon suspension injection and mitoxanone hydrochloride, both of which also suffer from significant local irritation during clinical use.

[0004] While there are examples of preparing tracers into liposomes in the existing technology, problems such as poor drug loading, poor encapsulation efficiency, or poor stability urgently need to be solved. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the issues of existing commercially available lymph node tracers, which often exhibit significant irritation, poor drug loading, or poor encapsulation efficiency or stability during clinical use, and thus require improvement. This invention provides a tracer liposome solution and its preparation method. The tracer liposome solution of this invention not only has high clinical recognition and minimal local irritation, but also exhibits rapid local dissipation after application, and can possess high drug loading, or high encapsulation efficiency and / or stability.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] This invention provides a tracer liposome solution, comprising a carrier liposome and an aqueous tracer solution encapsulated within the carrier liposome;

[0008] The carrier liposomes include phospholipids, cholesterol, and an encapsulating agent; the phospholipids include one or more of distearate phosphatidylcholine, hydrogenated soybean phospholipids, and sphingomyelin; and the tracer is one or more of methylene blue, indocyanine green, and mitoxantrone.

[0009] In this invention, in the tracer liposome solution, the phospholipids and cholesterol form a phospholipid bilayer, and the phospholipid bilayer and the encapsulating agent together form the carrier liposome.

[0010] In this invention, the carrier liposomes are generally the same as the blank liposome solution described in step S2 of the preparation method below.

[0011] In this invention, the carrier liposomes generally also contain water. This water is typically introduced during the dissolution of the encapsulating agent in the preparation method.

[0012] In this invention, the phospholipid is preferably distearate phosphatidylcholine and / or hydrogenated soybean phospholipid.

[0013] In this invention, the mass ratio of the phospholipid to the cholesterol can be (1-10):1, preferably (2-9):1, for example 3:1, 4:1, 5:1 or 7:1.

[0014] In this invention, the encapsulating agent may be ammonium sulfate and / or sucrose octasulfate, with ammonium sulfate being preferred.

[0015] In this invention, the tracer may be methylene blue, indocyanine green, or mitoxantrone, with methylene blue being preferred.

[0016] In this invention, the molar ratio of the tracer to the encapsulating agent can be ≥ (1:1), preferably ≥ (1.5:1), for example 2.0:1, 2.5:1 or 3:1.

[0017] In this invention, the water in the tracer aqueous solution is generally water for injection.

[0018] In this invention, the liposomes in the tracer liposome solution have a particle size of 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.

[0019] In this invention, the drug-to-liposome ratio in the tracer liposome solution can be ≥36%, preferably ≥42%, for example 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-to-liposome ratio is generally the ratio of tracer concentration (mg / mL) to phospholipid concentration (mg / mL) in the tracer liposome solution.

[0020] In some preferred embodiments, the drug-liposome ratio in the tracer liposome solution can be in the range of 42%-81%.

[0021] In this invention, the LPC content in the tracer liposome solution can be <1 mg / mL, preferably <0.76 mg / mL, for example 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.

[0022] In this invention, the total impurity content of related substances in the tracer liposome solution can be ≤0.95%, for example, 0.91%, 0.92% or 0.95%.

[0023] In this invention, the tracer content in the tracer liposome solution can be routinely adjusted while still meeting the requirements for visual identification of staining effects. The tracer content can be ≥2 mg / mL, preferably ≥4 mg / mL, more preferably ≥9 mg / mL, for example, 10.1 mg / mL or 10.3 mg / mL. The tracer content generally refers to the tracer concentration (mg) in 1 mL of the tracer liposome solution.

[0024] In this invention, the liposome encapsulation efficiency in the tracer liposome solution can be ≥98.9%, for example 99.2%, 99.3%, 99.4%, 99.5% or 99.6%.

[0025] This invention provides a method for preparing the tracer liposome solution, comprising the following steps:

[0026] S1. A phospholipid solution was injected into an internal aqueous phase solution, aged, extruded, and ultrafiltered to obtain a blank liposome solution.

[0027] The phospholipid solution comprises phospholipids, cholesterol, and a solvent; the phospholipids include one or more of distearylphosphatidylcholine (DSPC), hydrogenated soybean phospholipids (HSPC), and sphingomyelin (SM).

[0028] S2. The blank liposome solution is added to the tracer solution, mixed and encapsulated, and then ultrafiltered.

[0029] The encapsulation time is not less than 5 minutes, and the pH value of the system during encapsulation is 6-11.

[0030] In this invention, the tracer liposome solution is preferably as described above.

[0031] In S1, the phospholipid solution can be prepared by conventional methods in the art, such as dissolving phospholipids and cholesterol in a solvent, heating, and stirring to dissolve. The heating temperature can be no less than 60°C, for example, 63°C, 68°C, or 73°C.

[0032] In S1, the phospholipid solution contains preferably distearate phosphatidylcholine and / or hydrogenated soybean phospholipids.

[0033] In S1, the concentration of phospholipid in the phospholipid solution 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.

[0034] In S1, the mass ratio of phospholipids to cholesterol in the phospholipid solution can be (1-10):1, preferably (2-9):1, for example 3:1, 4:1, 5:1 or 7:1. The mass ratio of phospholipids to cholesterol in the phospholipid solution is generally the same as the mass ratio of phospholipids to cholesterol in the tracer liposomes.

[0035] In S1, the solvent in the phospholipid solution can be a conventional solvent in the art capable of dissolving phospholipids and cholesterol, such as anhydrous ethanol.

[0036] In S1, the volume ratio of the phospholipid solution to the internal aqueous phase solution can be 1:(2-20), preferably 1:5, 1:10 or 1:15.

[0037] In S1, the internal aqueous phase solution can be prepared by conventional methods in the art, such as dissolving the encapsulating agent in water, heating, and stirring to dissolve. The heating temperature can be no less than 60°C, for example, 63°C, 68°C, or 73°C.

[0038] In S1, the encapsulating agent in the internal aqueous phase solution may be ammonium sulfate and / or sucrose octasulfate, preferably ammonium sulfate.

[0039] In S1, the concentration of the encapsulating agent in the inner aqueous phase solution can be 0.2-1M, preferably 0.2-0.6M, for example 0.4M or 0.5M.

[0040] In S1, when the phospholipid solution is injected into the internal aqueous phase solution, the temperature of both solutions is generally not lower than 60°C, for example, 63°C, 68°C or 73°C.

[0041] In S1, those skilled in the art can conventionally adjust the injection time based on the volumes of the phospholipid solution and the inner aqueous phase solution. In a preferred embodiment, the injection time for injecting the phospholipid solution into the inner aqueous phase solution is 5 minutes.

[0042] In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the injection time can be 5-10 minutes.

[0043] In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the injection pore size can be conventional in the art, for example, 0.6 mm. The injection pore size generally refers to the fluid channel size when the phospholipid solution is injected into the inner aqueous phase through a syringe needle or a microfluidic chip channel.

[0044] In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the stirring speed of the inner aqueous phase solution can be conventional in the art, such as 300 rpm.

[0045] In S1, the ripening time is at least 5 minutes, can be 5 minutes to 2 hours, preferably 15 to 60 minutes, for example 30 minutes.

[0046] In S1, the ripening temperature can be 63℃-73℃, for example 63℃, 68℃ or 73℃.

[0047] In S1, during the ripening process, the rotation speed can be 150-250 rpm, for example, 200 rpm.

[0048] In S1, the extrusion operation and conditions can be conventional in the art, preferably extruding through a 200nm pore size polycarbonate membrane, followed by extrusion through an 80nm pore size polycarbonate membrane. During the extrusion process, the number of membrane layers and the number of extrusions can be conventionally adjusted according to the particle size of the finished product.

[0049] In a preferred embodiment, a 200nm*3 (layer) polycarbonate film is first extruded once, and then an 80nm*3 (layer) polycarbonate film is extruded three times, resulting in an average particle size of 70nm for the finished product.

[0050] In a preferred embodiment, an 80nm*3 (layer) polycarbonate film is extruded twice to achieve an average particle size of 80nm.

[0051] In a preferred embodiment, a 200nm*3 (layer) polycarbonate film is first extruded once, and then an 80nm*3 (layer) polycarbonate film is extruded once, resulting in an average particle size range of 131nm for the finished product.

[0052] In a preferred embodiment, a 200nm*3 (layer) polycarbonate film is first extruded once, and then an 80nm*1 (layer) polycarbonate film is extruded once, resulting in an average particle size of 180nm for the finished product.

[0053] In a preferred embodiment, a 200nm*3 (layer) polycarbonate film is extruded once, and the resulting product has an average particle size of 200nm.

[0054] In S1, after extrusion and before ultrafiltration, the system can be cooled to ≤25℃.

[0055] In S1, the ultrafiltration operation and conditions can be conventional in the art, generally using room temperature water for injection as the external aqueous phase for ultrafiltration. The purpose of ultrafiltration is generally to remove unencapsulated encapsulating agent to provide a concentration gradient for subsequent encapsulation operations.

[0056] In S1, the ultrafiltration can be performed using a 300KD ultrafiltration membrane for constant volume concentration ultrafiltration.

[0057] In S1, the number of ultrafiltration cycles can be conventional in the art, for example, ≥8 times.

[0058] In step S2, the actual detection concentration of the encapsulating agent in the blank liposome solution can be 0.04-0.09 M, for example, 0.05 M, 0.0625 M, or 0.0833 M. The actual detection concentration of the encapsulating agent refers to the concentration of the remaining encapsulating agent in the blank liposome solution after ultrafiltration to remove the unencapsulated encapsulating agent in step S1, i.e., the actual content of the encapsulating agent in the blank liposome solution when mixed with the tracer solution.

[0059] In S2, the tracer solution can be prepared by conventional methods in the art, such as mixing the tracer and solvent and heating to dissolve them. The heating and dissolving temperature can be 35-45°C, for example, 40°C.

[0060] In S2, when the blank liposome solution is added to the tracer solution, the temperature of the tracer solution can be 30-40°C, for example, 35°C.

[0061] In S2, the tracer solution contains one or more of methylene blue, indocyanine green, and mitoxantrone, such as methylene blue, indocyanine green, or mitoxantrone, with methylene blue being preferred.

[0062] In S2, the solvent in the tracer solution can be water for injection.

[0063] In S2, the tracer concentration in the tracer solution may not exceed 3% by mass, for example, 1.48%.

[0064] In step S2, the molar ratio of the tracer in the tracer solution to the encapsulating agent actually detected in the blank liposome solution can be ≥ (1:1), preferably ≥ (1.5:1), for example, 2.0:1, 2.5:1, or 3:1. The encapsulating agent actually detected refers to the encapsulating agent remaining in the blank liposome solution after the unencapsulated encapsulating agent is removed by ultrafiltration in step S1. When the above molar ratio is ≤ 2:1, the above molar ratio is generally the same as the molar ratio of tracer to encapsulating agent in the tracer liposome solution. When the above molar ratio is > 2:1, excess tracer will be partially removed during the ultrafiltration process in S2, and the molar ratio of tracer to encapsulating agent in the tracer liposome solution will generally be slightly greater than 2:1.

[0065] In S2, based on the actual content of the encapsulating agent detected in the blank liposome solution and the controlled molar ratio of tracer to encapsulating agent, those skilled in the art can routinely adjust the amount of tracer solution added.

[0066] In step S2, those skilled in the art can conventionally adjust the mixing time of the blank liposome solution and the tracer solution based on their volumes. In a preferred embodiment, the blank liposome solution is added to the tracer solution and mixed for 5 minutes, then the pH of the system is adjusted for encapsulation. The pH of the system is typically adjusted using hydrochloric acid or sodium hydroxide solution (concentration can be 2M).

[0067] In step S2, the pH value of the mixture of the blank liposome solution and the tracer solution is adjusted to the encapsulation pH value and the temperature is adjusted to the encapsulation temperature before encapsulation begins.

[0068] In S2, the encapsulation temperature may be no less than 55°C, preferably 60-76°C, more preferably 65-70°C, for example 68°C.

[0069] In S2, the encapsulation time is preferably 5 min-60 min, for example 10-55 min, such as 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min or 50 min.

[0070] In S2, during the encapsulation process, the pH value of the system is preferably 6.5-10.5, for example 7, 7.5, 8, 8.5, 9, 9.8, 10, 10.5 or 11.

[0071] In S2, after encapsulation, the temperature is generally reduced to ≤25℃.

[0072] In S2, the operation and conditions of ultrafiltration can be conventional in the art. In this invention, the purpose of ultrafiltration is generally to remove incompletely encapsulated drugs and to provide an isotonic and pH-similar external environment for the human body.

[0073] In S2, the ultrafiltrate for ultrafiltration can be a mixed aqueous solution of glucose and HEPES, a mixed aqueous solution of sodium dihydrogen phosphate and glucose, or a mixed aqueous solution of sodium chloride and HEPES.

[0074] In the glucose and HEPES mixed aqueous solution, the concentration of HEPES can be 6-8 mg / mL, for example 4.05 mg / mL. The concentration of glucose can be 40-50 mg / mL, for example 47 mg / mL.

[0075] In the aqueous solution of sodium dihydrogen phosphate and glucose, the concentration of sodium dihydrogen phosphate can be 6-8 mg / mL, for example, 6.8 mg / mL. The concentration of glucose can be 40-50 mg / mL, for example, 47 mg / mL.

[0076] In the mixed aqueous solution of sodium chloride and HEPES, the concentration of sodium chloride can be 5-12 mg / mL, for example 8.45 mg / mL. The concentration of HEPES can be 3-5 mg / mL, for example 4.1 mg / mL.

[0077] In S2, the pH value of the ultrafiltrate can be 2-11, preferably 3-10, for example 4, 6.5, 7, 8 or 9.

[0078] In S2, the ultrafiltration can be performed using a 300KD ultrafiltration membrane for constant volume concentration ultrafiltration.

[0079] In S2, the number of ultrafiltration cycles can be ≥10.

[0080] The present invention also provides a tracer liposome solution prepared by the preparation method described above.

[0081] In this invention, the liposomes in the tracer liposome solution have a particle size of 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.

[0082] This invention provides a method for preparing a tracer liposome solution, comprising the following steps:

[0083] S1. A phospholipid solution was injected into an internal aqueous phase solution, aged, extruded, and ultrafiltered to obtain a blank liposome solution.

[0084] The phospholipid solution comprises phospholipids, cholesterol, and a solvent; the phospholipids include one or more of distearate phosphatidylcholine (DSPC), hydrogenated soybean phospholipids (HSPC), and sphingomyelin (SM); and the maturation time is at least 5 minutes.

[0085] S2. The blank liposome solution is added to the tracer solution, mixed and encapsulated, and then ultrafiltered.

[0086] In S1, the phospholipid solution can be prepared by conventional methods in the art, such as dissolving phospholipids and cholesterol in a solvent, heating, and stirring to dissolve. The heating temperature can be no less than 60°C, for example, 63°C, 68°C, or 73°C.

[0087] In S1, the phospholipid solution contains preferably distearate phosphatidylcholine and / or hydrogenated soybean phospholipids.

[0088] In S1, the concentration of phospholipids in the phospholipid solution can be 237-711 mg / mL, preferably 400-800 mg / mL, for example 423 mg / mL, 474 mg / mL, 500 mg / mL, 708 mg / mL, or 750 mg / mL. The mass ratio of phospholipids to cholesterol in the phospholipid solution is generally the same as the mass ratio of phospholipids to cholesterol in the tracer liposomes.

[0089] In S1, the mass ratio of phospholipids to cholesterol in the phospholipid solution can be (1-10):1, preferably (2-9):1, for example 3:1, 4:1, 5:1 or 7:1.

[0090] In S1, the solvent in the phospholipid solution can be a conventional solvent in the art capable of dissolving phospholipids and cholesterol, such as anhydrous ethanol.

[0091] In S1, the volume ratio of the phospholipid solution to the internal aqueous phase solution can be 1:(2-20), preferably 1:5, 1:10 or 1:15.

[0092] In S1, the internal aqueous phase solution can be prepared by conventional methods in the art, such as dissolving the encapsulating agent in water, heating, and stirring to dissolve. The heating temperature can be no less than 60°C, for example, 63°C, 68°C, or 73°C.

[0093] In S1, the encapsulating agent in the internal aqueous phase solution may be ammonium sulfate and / or sucrose octasulfate, preferably ammonium sulfate.

[0094] In S1, the concentration of the encapsulating agent in the inner aqueous phase solution can be 0.2-1M, preferably 0.2-0.6M, for example 0.4M or 0.5M.

[0095] In S1, when the phospholipid solution is injected into the internal aqueous phase solution, the temperature of both solutions is generally not lower than 60°C, for example, 63°C, 68°C or 73°C.

[0096] In S1, those skilled in the art can conventionally adjust the injection time based on the volumes of the phospholipid solution and the inner aqueous phase solution. In a preferred embodiment, the injection time for injecting the phospholipid solution into the inner aqueous phase solution is 5 minutes.

[0097] In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the injection time can be 5-10 minutes.

[0098] In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the injection pore size can be conventional in the art, for example, 0.6 mm. The injection pore size generally refers to the fluid channel size when the phospholipid solution is injected into the inner aqueous phase through a syringe needle or a microfluidic chip channel.

[0099] In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the stirring speed of the inner aqueous phase solution can be conventional in the art, such as 300 rpm.

[0100] In S1, the curing time can be 5 min to 2 h, preferably 15 to 60 min, for example 30 min.

[0101] In S1, the ripening temperature can be 63℃-73℃, for example 63℃, 68℃ or 73℃.

[0102] In S1, during the ripening process, the rotation speed can be 150-250 rpm, for example, 200 rpm.

[0103] In S1, the extrusion operation and conditions can be conventional in the art, preferably extruding through a 200nm pore size polycarbonate membrane, followed by extrusion through an 80nm pore size polycarbonate membrane. During the extrusion process, the number of membrane layers and the number of extrusions can be conventionally adjusted according to the particle size of the finished product.

[0104] In a preferred embodiment, a 200nm*3 (layer) polycarbonate film is first extruded once, and then an 80nm*3 (layer) polycarbonate film is extruded three times, resulting in an average particle size of 70nm for the finished product.

[0105] In a preferred embodiment, an 80nm*3 (layer) polycarbonate film is extruded twice to achieve an average particle size of 80nm.

[0106] In a preferred embodiment, a 200nm*3 (layer) polycarbonate film is first extruded once, and then an 80nm*3 (layer) polycarbonate film is extruded once, resulting in an average particle size range of 131nm for the finished product.

[0107] In a preferred embodiment, a 200nm*3 (layer) polycarbonate film is first extruded once, and then an 80nm*1 (layer) polycarbonate film is extruded once, resulting in an average particle size of 180nm for the finished product.

[0108] In a preferred embodiment, a 200nm*3 (layer) polycarbonate film is extruded once, and the resulting product has an average particle size of 200nm.

[0109] In S1, after extrusion and before ultrafiltration, the system can be cooled to ≤25℃.

[0110] In S1, the ultrafiltration operation and conditions can be conventional in the art, generally using room temperature water for injection as the external aqueous phase for ultrafiltration. The purpose of ultrafiltration is generally to remove unencapsulated encapsulating agent to provide a concentration gradient for subsequent encapsulation operations.

[0111] In S1, the ultrafiltration can be performed using a 300KD ultrafiltration membrane for constant volume concentration ultrafiltration.

[0112] In S1, the number of ultrafiltration cycles can be conventional in the art, for example, ≥8 times.

[0113] In step S2, the actual detection concentration of the encapsulating agent in the blank liposome solution can be 0.04-0.09 M, for example, 0.05 M, 0.0625 M, or 0.0833 M. The actual detection concentration of the encapsulating agent refers to the concentration of the remaining encapsulating agent in the blank liposome solution after ultrafiltration to remove the unencapsulated encapsulating agent in step S1, i.e., the actual content of the encapsulating agent in the blank liposome solution when mixed with the tracer solution.

[0114] In S2, the tracer solution can be prepared by conventional methods in the art, such as mixing the tracer and solvent and heating to dissolve them. The heating and dissolving temperature can be 35-45°C, for example, 40°C.

[0115] In S2, when the blank liposome solution is added to the tracer solution, the temperature of the tracer solution can be 30-40°C, for example, 35°C.

[0116] In S2, the tracer solution contains one or more of methylene blue, indocyanine green, and mitoxantrone, such as methylene blue, indocyanine green, or mitoxantrone, with methylene blue being preferred.

[0117] In S2, the solvent in the tracer solution can be water for injection.

[0118] In S2, the tracer concentration in the tracer solution may not exceed 3% by mass, for example, 1.48%.

[0119] In step S2, the molar ratio of the tracer in the tracer solution to the encapsulating agent actually detected in the blank liposome solution can be ≥ (1:1), preferably ≥ (1.5:1), for example, 2.0:1, 2.5:1, or 3:1. The encapsulating agent actually detected refers to the encapsulating agent remaining in the blank liposome solution after the unencapsulated encapsulating agent is removed by ultrafiltration in step S1. When the above molar ratio is ≤ 2:1, the above molar ratio is generally the same as the molar ratio of tracer to encapsulating agent in the tracer liposome solution. When the above molar ratio is > 2:1, excess tracer will be partially removed during the ultrafiltration process in S2, and the molar ratio of tracer to encapsulating agent in the tracer liposome solution will generally be slightly greater than 2:1.

[0120] In S2, based on the actual content of the encapsulating agent detected in the blank liposome solution and the controlled molar ratio of tracer to encapsulating agent, those skilled in the art can routinely adjust the amount of tracer solution added.

[0121] In step S2, those skilled in the art can conventionally adjust the mixing time of the blank liposome solution and the tracer solution based on their volumes. In a preferred embodiment, the blank liposome solution is added to the tracer solution and mixed for 5 minutes, then the pH of the system is adjusted for encapsulation. The pH of the system is typically adjusted using hydrochloric acid or sodium hydroxide solution (concentration can be 2M).

[0122] In step S2, the pH value of the mixture of the blank liposome solution and the tracer solution is adjusted to the encapsulation pH value and the temperature is adjusted to the encapsulation temperature before encapsulation begins.

[0123] In S2, the encapsulation temperature may be no less than 55°C, preferably 60-76°C, more preferably 65-70°C, for example 68°C.

[0124] In S2, the encapsulation time may be no less than 5 minutes, preferably 5 minutes to 60 minutes, for example 10 to 55 minutes, such as 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes or 50 minutes.

[0125] In S2, during the encapsulation process, the pH value of the system can be 6-11, preferably 6.5-10.5, such as 7, 7.5, 8, 8.5, 9, 9.8, 10, 10.5 or 11.

[0126] In S2, after encapsulation, the temperature is generally reduced to ≤25℃.

[0127] In S2, the operation and conditions of ultrafiltration can be conventional in the art. In this invention, the purpose of ultrafiltration is generally to remove incompletely encapsulated drugs and to provide an isotonic and pH-similar external environment for the human body.

[0128] In S2, the ultrafiltrate for ultrafiltration can be a mixed aqueous solution of glucose and HEPES, a mixed aqueous solution of sodium dihydrogen phosphate and glucose, or a mixed aqueous solution of sodium chloride and HEPES.

[0129] In the glucose and HEPES mixed aqueous solution, the concentration of HEPES can be 6-8 mg / mL, for example 4.05 mg / mL. The concentration of glucose can be 40-50 mg / mL, for example 47 mg / mL.

[0130] In the aqueous solution of sodium dihydrogen phosphate and glucose, the concentration of sodium dihydrogen phosphate can be 6-8 mg / mL, for example, 6.8 mg / mL. The concentration of glucose can be 40-50 mg / mL, for example, 47 mg / mL.

[0131] In the mixed aqueous solution of sodium chloride and HEPES, the concentration of sodium chloride can be 5-12 mg / mL, for example 8.45 mg / mL. The concentration of HEPES can be 3-5 mg / mL, for example 4.1 mg / mL.

[0132] In S2, the pH value of the ultrafiltrate can be 2-11, preferably 3-10, for example 4, 6.5, 7, 8 or 9.

[0133] In S2, the ultrafiltration can be performed using a 300KD ultrafiltration membrane for constant volume concentration ultrafiltration.

[0134] In S2, the number of ultrafiltration cycles can be ≥10.

[0135] The present invention also provides a tracer liposome solution prepared by the preparation method described above.

[0136] In this invention, the tracer liposome solution contains tracer liposomes with a particle size of 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.

[0137] In this invention, the drug-to-liposome ratio in the tracer liposome solution can be ≥36%, preferably ≥42%, for example 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-to-liposome ratio is generally the ratio of tracer concentration (mg / mL) to phospholipid concentration (mg / mL) in the tracer liposome solution.

[0138] In some preferred embodiments, the drug-liposome ratio in the tracer liposome solution can be in the range of 42%-81%.

[0139] In this invention, the LPC content in the tracer liposome solution can be <1 mg / mL, preferably <0.76 mg / mL, for example 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.

[0140] In this invention, the total impurity content of related substances in the tracer liposome solution can be ≤0.95%, for example, 0.91%, 0.92% or 0.95%.

[0141] In this invention, the tracer content in the tracer liposome solution can be routinely adjusted while still meeting the requirements for visual identification of staining effects. The tracer content can be ≥2 mg / mL, preferably ≥4 mg / mL, more preferably ≥9 mg / mL, for example, 10.1 mg / mL or 10.3 mg / mL. The tracer content generally refers to the tracer concentration (mg) in 1 mL of the tracer liposome solution.

[0142] In this invention, the liposome encapsulation efficiency in the tracer liposome solution can be ≥98.9%, for example 99.2%, 99.3%, 99.4%, 99.5% or 99.6%.

[0143] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0144] The reagents and raw materials used in this invention are all commercially available.

[0145] The positive and progressive effects of this invention are as follows:

[0146] The tracer liposome solution of the present invention not only has high clinical recognition, low local irritation, and rapid local dissipation after use, but also has a high drug loading capacity, or a high encapsulation rate and / or stability.

[0147] In some preferred embodiments, the tracer liposomes of the present invention not only have a high drug loading capacity, but also a high encapsulation efficiency and / or stability. Attached Figure Description

[0148] Figure 1 shows the tracing images of rat lymph nodes with liposome solutions of different particle sizes. In Figure 1, the particle sizes of the finished products in parts A to C are 70 nm (Example 59), 200 nm (Example 60), and 131 nm (Example 1), respectively.

[0149] Figure 2 shows rat tracing images with different encapsulating agents. Part A of Figure 2 shows the encapsulation of ammonium sulfate as the inner aqueous phase in Example 1, and Part B of Figure 2 shows the encapsulation of sucrose octasulfate as the inner aqueous phase in Example 62.

[0150] Figures 3 and 4 show a comparison of rat lymph node staining after encapsulation with different staining agents using the same process. In Figure 3, part A shows the mitoxantrone liposome solution prepared in Example 63; parts B of Figure 3 and Part B of Figure 4 show the methylene blue liposome solution prepared in Example 1; and part A of Figure 4 shows the indocyanine green liposome solution prepared in Example 64.

[0151] Figure 5 shows a comparison of rat lymph node tracing using commercially available methylene blue injection and methylene blue liposome solution from Example 1. In this figure, 1 represents commercially available methylene blue injection, and 2 represents methylene blue liposome solution from Example 1.

[0152] Figure 6 shows a comparison of the effects of commercially available methylene blue injection, mitoxantrone hydrochloride injection, and methylene blue liposome solution from Example 1 on subcutaneous lymphatic stimulation in rats. In the figures, 1 represents commercially available methylene blue injection, 2 represents methylene blue liposome solution from Example 1, and 3 represents commercially available mitoxantrone hydrochloride injection.

[0153] Figure 7 shows a comparison of the irritation differences between the methylene blue liposome solution of Example 1 and the commercially available nano-carbon suspension injection. In this figure, 1 represents the commercially available nano-carbon suspension injection, and 2 represents the methylene blue liposome solution of Example 1.

[0154] Figure 8 shows photographs of the free drug in the product after encapsulation with methylene blue and ammonium sulfate at different molar ratios (without ultrafiltration in step S2) passing through a gel column (the intermediate encapsulation efficiency was tested during gel column encapsulation). In Figure 8, the molar ratios of methylene blue and ammonium sulfate in parts A to C are 2.5:1 (Example 17), 2.0:1 (Example 1), and 1.5:1 (Example 16), respectively. Detailed Implementation

[0155] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the concept and scope of the invention.

[0156] In the following examples or comparative examples, the nano-carbon suspension injection (trade name: Canalin) was purchased from Chongqing Laimi Pharmaceutical Co., Ltd.; mitoxantrone hydrochloride (trade name: Futasu) was purchased from Shenzhen CR Nine Ventures Pharmaceutical Co., Ltd.; and methylene blue injection was purchased from Jichuan Pharmaceutical Group Co., Ltd.

[0157] In this invention:

[0158] The content of phospholipid degradation impurities in the finished product (finished product LPC content) generally refers to the content (mg) of phospholipid degradation impurities in liposome solution (1 mL).

[0159] The phospholipid degradation impurity content (LPC content in blank liposome solution) is the LPC content (mg) in 1 mL of the blank liposome solution obtained after ultrafiltration in step S1. The specific test method is the same as that for the LPC content detection of the finished product.

[0160] The LPC content of a 0-day finished product generally refers to the LPC content of the liposome solution prepared in the experiment without any treatment and directly tested.

[0161] The LPC content of the finished liposome solution, which is influencing factors at 40℃, generally refers to the LPC content tested after the finished liposome solution is sealed and stored at 40℃ and 75% RH (75% humidity) for 14 days.

[0162] The tracer content generally refers to the tracer content (mg) in 1 mL of liposome solution.

[0163] The 0-day encapsulation rate generally refers to the encapsulation rate of the liposome solution prepared in the experiment without any treatment and directly tested and calculated.

[0164] The encapsulation rate in the encapsulation process refers to the encapsulation rate of the intermediate, that is, the encapsulation rate of the liposome solution before ultrafiltration in step S2.

[0165] The particle size of the finished product at 0 days generally refers to the particle size of the liposome solution prepared in the experiment without any treatment and directly measured.

[0166] Test method for physical stability at 0 days: Take 0.5 mL of liposome solution (the liposome solution prepared in the experiment was not treated in any way and was directly tested) and centrifuge (10000 rpm for 5 min) and observe. If no obvious precipitation is visible to the naked eye, it indicates physical stability; if solid is visible at the bottom, the solid precipitate is the sediment. A very small amount of sediment indicates relatively stable physical stability, while a large amount of sediment indicates physical instability.

[0167] "Encapsulation rate of factors affecting 40℃" refers to the encapsulation rate of liposome solutions after being packaged in conventional vials and sealed with a cap, and stored at 40℃ and an ambient humidity of 75%RH (75% humidity) for 14 days.

[0168] The drug-to-liposome ratio generally refers to the ratio of tracer concentration (mg / ml) to phospholipid concentration (mg / ml) in the finished tracer liposome solution. The tracer concentration can be detected by the method for detecting methylene blue content in item "2" of Example 1, and the phospholipid concentration can be detected by the method for detecting phospholipid degradation impurities in item "1" of Example 1.

[0169] Example 1: Preparation of methylene blue liposome solution

[0170] S1 involves injecting the phospholipid solution into the internal aqueous phase solution, maturing, pressing, and then ultrafiltration in a single step to obtain a blank liposome solution; wherein:

[0171] Preparation of phospholipid solution: Dissolve 63.14g DSPC and 21.05g cholesterol in 133ml anhydrous ethanol and incubate in a water bath at 68℃±5℃. The phospholipid concentration in the solution is 474mg / mL. The mass ratio of phospholipid to cholesterol is 3:1.

[0172] Preparation of the internal aqueous phase solution: Dissolve 88.01 g of ammonium sulfate in 1333 g of water for injection and heat to 68 °C. The volume ratio of the phospholipid solution to the internal aqueous phase solution is 1:10, and the concentration of the ammonium sulfate solution is 0.5 M.

[0173] Emulsion injection: Maintain the solution temperature at 68℃±2℃, slowly inject the phospholipid solution into the inner aqueous phase solution, with an injection orifice diameter of 0.6mm, an inner aqueous phase stirring speed of 300rpm, and an injection time of approximately 5min.

[0174] Maturation: After injection, maintain the emulsion at 68℃±5℃ for 30 minutes at a speed of 200 rpm.

[0175] Extrusion: First, extrude once using a 200nm*3 polycarbonate membrane, then extrude once using an 80nm*3 polycarbonate membrane. The average particle size range of the resulting product is 100-150nm. After extrusion, the sample is cooled to ≤25℃ and then ultrafiltered.

[0176] One-time ultrafiltration: using room temperature water for injection as the external aqueous phase, constant volume concentration ultrafiltration was performed using a 300KD ultrafiltration membrane, and the number of ultrafiltration cycles was 8.

[0177] S2: After adding the blank liposome solution to the API solution and mixing for 5 min, the pH was adjusted to 9.8 with 2M sodium hydroxide solution. The temperature was then rapidly increased to 68℃ and maintained for 15 min (encapsulation was performed immediately after pH adjustment, i.e., encapsulation pH was 9.8, encapsulation temperature was 68℃, and encapsulation time was 15 min). The temperature was then rapidly decreased to ≤25℃, and a second ultrafiltration was performed to obtain the methylene blue liposome solution. Wherein:

[0178] API solution preparation: Take 40g of methylene blue (calculated as anhydrous), add 2660g of water for injection, heat to 40℃ to dissolve, and after dissolution, cool down to maintain the solution temperature at 35℃. The mass percentage concentration of methylene blue in the API solution is 1.48%.

[0179] Based on the actual ammonium sulfate content detected in the blank liposome solution and the controlled molar ratio of methylene blue to ammonium sulfate, the amount of API solution added was adjusted. In Example 1, during the mixing and encapsulation process, the actual concentration of ammonium sulfate in the blank liposome solution was 0.0625 M, and the molar ratio of methylene blue to ammonium sulfate was set to 2:1, allowing for further calculation of the methylene blue solution volume.

[0180] Preparation of secondary ultrafiltrate: The ultrafiltrate is a mixed aqueous solution of glucose and HEPES. Take 4.7g of glucose and 0.405g of HEPES, add water and stir to dissolve, then dilute to 100ml and adjust the pH to 6.5 with sodium hydroxide.

[0181] Secondary ultrafiltration: Using room temperature secondary ultrafiltrate as the external aqueous phase, constant volume concentration ultrafiltration is performed using a 300KD ultrafiltration membrane, and the ultrafiltration is performed 10 times.

[0182] Example 1: Stability test of methylene blue liposome solution

[0183] The experiment was repeated three times according to the method in Example 1, yielding batches 1-3 of methylene blue liposome solutions. The three batches of methylene blue liposome solutions were then tested according to the methods described below.

[0184] 1. Under accelerated conditions of 25℃ and 75%RH (75% humidity), the test solutions of samples at each time point were prepared and the phospholipid degradation impurities (LPC) were detected as follows: 1 ml of liposome solution was accurately measured into a 5 ml volumetric flask, diluted to the mark with diluent (methanol), shaken well, centrifuged at 10000 rpm for 10 min, and the sample was injected for detection according to the table below.

[0185] Table 1 Test Conditions

[0186] The test results are shown in the table below.

[0187] Table 2. LPC content (mg / ml) of finished product after accelerated testing at 25℃ and 75% RH.

[0188] 2. At 25℃ and under accelerated conditions of 75%RH (75% humidity), the samples at each time point were prepared into test solutions and the methylene blue content and impurity content were detected as follows.

[0189] Accurately measure 1 ml of liposome solution into a 100 ml volumetric flask, add 9 ml of methanol to break the emulsion, and finally dilute to the mark with diluent (25% acetonitrile solution). Shake well, centrifuge at 10000 rpm for 10 min, and inject the sample according to the table below.

[0190] Table 3 Test Conditions

[0191] The test results are shown in the table below.

[0192] Table 4. Total Related Substances in Accelerated Test at 25℃ and 75% RH

[0193] Table 5. Methylene Blue Content in Accelerated Test at 25℃ and 75% RH

[0194] 3. Under accelerated conditions of 25℃ and 75%RH (75% humidity), the encapsulation efficiency of samples at each time point was calculated as follows.

[0195] According to the test solution and detection method in "2. Detection of Methylene Blue Content and Impurity Content" above, 100 μL of the test sample was passed through a gel column to separate free and liposomes. After the concentrations of free and liposomes were determined according to the content method, the encapsulation efficiency was calculated as: Encapsulation efficiency = Liposome content / (Free content + Liposome content). The calculation results are shown in the table below.

[0196] Table 6 Encapsulation efficiency in accelerated testing at 25℃ and 75% RH

[0197] 4. Under accelerated conditions of 25℃ and 75%RH (75% humidity), the particle size of the samples at each time point was determined as follows.

[0198] Take 10 μL of the test solution from "2. Determination of methylene blue content and impurity content" above, add 10 mL of pure water and mix well. Then, take a sample and use a dynamic light scattering particle size analyzer to detect the particle size. The particle size results are shown in Table 7.

[0199] Table 7 Particle size in accelerated test at 25℃ and 75% RH

[0200] Unless otherwise specified, the methods for testing the LPC content, total related substances, methylene blue content, encapsulation efficiency, and particle size of the liposome solution in this invention are the same as those in Example 1.

[0201] Examples 2-3 and Comparative Examples 1-2: Phospholipid Types

[0202] In Examples 2-3 and Comparative Examples 1-2, the types of phospholipids are shown in the table below. Other operations and conditions are the same as in Example 1. Liposome solutions were prepared and the test results are shown in the table below.

[0203] Table 8 Screening of Phospholipid Types

[0204] In the table, all test data are for the final product liposome solution. "NA" indicates that the data could not be tested after sedimentation.

[0205] Therefore, when the phospholipid is DSPC or HSPC, the encapsulation efficiency is higher and the stability is better.

[0206] Example 4-5 Phospholipid to Cholesterol Mass Ratio

[0207] In Examples 4-5, the phospholipid:cholesterol mass ratios were 1:1 and 10:1, respectively. All other operations and conditions were the same as in Example 1. Liposome solutions were prepared, and the test results are shown in the table below.

[0208] Table 9. Investigation of the mass ratio of phospholipids and cholesterol

[0209] In Table 9, apart from the encapsulation rate during the encapsulation process, all other test data are test data for the final product liposome solution.

[0210] This shows that when the mass ratio of phospholipids to cholesterol is 3-5:1, the encapsulation efficiency is higher and the stability is better.

[0211] Volume ratio of phospholipid solution and internal aqueous phase solution in Examples 6-7

[0212] In Examples 6 and 7, the volume ratio of phospholipid solution to internal aqueous phase solution was 1:2 and 1:20, respectively. Other operations and conditions were the same as in Example 1. Liposome solutions were prepared, and the test results are shown in the table below.

[0213] Table 10 Investigation of the volume ratio of phospholipid solution and internal aqueous phase solution

[0214] All test data in the table are for the final product liposome solution.

[0215] Therefore, when the volume ratio of phospholipid solution to internal aqueous solution is 1:(2-20), the encapsulation efficiency and stability of the obtained product meet the requirements. When the ratio is 1:(2-10), the encapsulation efficiency is higher and the stability is better.

[0216] Examples 8-9 Phospholipid Concentration

[0217] In Examples 8 and 9, the amounts of anhydrous ethanol used in the phospholipid solutions were 266 mL and 89 mL, respectively, and the concentrations of phospholipids in the solutions were 237 mg / mL and 708 mg / mL, respectively. All other operations and conditions were the same as in Example 1. Liposome solutions were prepared, and the test results are shown in the table below.

[0218] Table 11. Investigation of phospholipid concentration

[0219] All test data are for the final product liposome solution.

[0220] Therefore, it can be seen that liposome solutions have better encapsulation efficiency and stability in the phospholipid solution concentration range of 200-800 mg / mL.

[0221] Examples 10-11 Encapsulating agent concentration

[0222] In Examples 10-11, the preparation and concentration of ammonium sulfate solution are shown in the table below. Other operations and conditions are the same as in Example 1. Liposome solutions were prepared and the test results are shown in the table below.

[0223] Table 12 Investigation of Encapsulating Agent Concentration

[0224] The test data in the table are all test data of the final product liposome solution.

[0225] Therefore, it can be seen that liposome solutions have better encapsulation efficiency and stability in the ammonium sulfate solution range of 0.2-1M, with the best effect at 0.2-0.6M.

[0226] Examples 12-15: Curing Time

[0227] In Comparative Examples 3 and Examples 12-15, the maturation times are shown in the table below. Other operations and conditions are the same as in Example 1. Liposome solutions were prepared and the test results are shown in the table below.

[0228] Table 13. Assessment of maturation time

[0229] Therefore, it can be seen that with a maturation time of 15-60 minutes, the blank liposome solution is more stable, and the finished liposome solution has good physical stability.

[0230] Examples 16-17: Molar ratio of methylene blue and ammonium sulfate

[0231] In Examples 16-17, the actual concentration of ammonium sulfate, the molar ratio of methylene blue to ammonium sulfate in the blank liposome solution during the encapsulation process are shown in the table below. Other operations and conditions are the same as in Example 1. Liposome solutions were prepared and the test results are shown in the table below.

[0232] Table 14 Investigation of the molar ratio of methylene blue and ammonium sulfate

[0233] Therefore, 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 and the stability is better.

[0234] Examples 18-53: Effects of Encapsulation Time, Temperature, and pH

[0235] In Examples 18-53, the encapsulation time, temperature, and pH value are shown in the table below. Other operations and conditions are the same as in Example 1. Liposome solutions were prepared and the test results are shown in the table below.

[0236] Table 15 Effects of Encapsulation Time, Temperature, and pH

[0237] The data in the table are all test results for the finished liposome solution.

[0238] Therefore, the drug-to-ester ratio in the liposome solutions of this invention is greater than 42%. Under the same encapsulation temperature and time, the drug-to-ester ratio in the liposome solutions is even higher when the pH value is between 9 and 11.

[0239] Examples 54-58 External aqueous phase pH values

[0240] In Examples 54-58, the pH value of the external aqueous phase was adjusted using the methods shown in the table below. Other operations and conditions were the same as in Example 1. Liposome solutions were prepared and the test results are shown in the table below.

[0241] Table 16. Investigation of pH values ​​in external aqueous phases

[0242] The data in the table are all test results of the finished liposome solution.

[0243] Therefore, it can be concluded that liposome solutions are more stable when the pH of the external aqueous phase is between 4 and 9.

[0244] Example 59

[0245] The only difference from Example 1 is the extrusion process: first, a 200nm*3 polycarbonate film is used for extrusion once, and then an 80nm*3 polycarbonate film is used for extrusion three times. The average particle size of the resulting product is 70nm. All other operations and conditions are the same as in Example 1, and a liposome solution is prepared.

[0246] Example 60

[0247] The only difference from Example 1 is the extrusion process: a 200nm*3 polycarbonate film is used for one extrusion, and the average particle size of the resulting product is 200nm. All other operations and conditions are the same as in Example 1, and a liposome solution is prepared.

[0248] Example 61

[0249] The only difference from Example 1 is the extrusion process: an 80nm*3 polycarbonate film is used for extrusion twice, and the average particle size of the resulting product is 80nm. All other operations and conditions are the same as in Example 1, and a liposome solution is prepared.

[0250] Example 62

[0251] The only difference from Example 1 is that: first, a 200nm*3 polycarbonate film is used for extrusion once, and then an 80nm*1 polycarbonate film is used for extrusion once. The average particle size of the resulting product is 180nm. All other operations and conditions are the same as in Example 1, and a liposome solution is prepared.

[0252] Example 63

[0253] In Example 1, ammonium sulfate was replaced with an equal amount of sucrose octasulfate, while other operations and conditions remained unchanged, to prepare a liposome solution.

[0254] Example 64

[0255] In Example 1, methylene blue was replaced with an equal amount of mitoxantrone, and all other operations and conditions were the same as in Example 1 to prepare a mitoxantrone liposome solution.

[0256] Example 65

[0257] In Example 1, methylene blue was replaced with an equal amount of indocyanine green, and all other operations and conditions were the same as in Example 1, to prepare an indocyanine green liposome solution.

[0258] The drug-liposome ratio and LPC content of the finished product at day 0 for different tracer liposome solutions are shown in Table 17 below.

[0259] Table 17 Effects of different tracer liposomes

[0260] Therefore, in terms of drug-liposome ratio and LPC content in the finished product at day 0, the methylene blue liposome in Example 1 is the best, followed by the indocyanine green liposome in Example 65 and the mitoxantrone liposome in Example 64.

[0261] Example 66

[0262] The secondary ultrafiltrate in Example 1 was replaced with a mixed aqueous solution of sodium chloride and HEPES, and all other operations and conditions were the same as in Example 1.

[0263] The mixed aqueous solution was prepared as follows: 421g of sodium chloride and 202.5g of HEPES were added to 49.4kg of water for injection. After stirring to dissolve, the pH was adjusted to 6.5 with sodium hydroxide, and the solution was stirred for 5 minutes before use. In the ultrafiltrate, the concentration of sodium chloride was 8.45mg / mL, and the concentration of HEPES was 4.1mg / mL.

[0264] Example 67

[0265] The secondary ultrafiltration solution in Example 1 was replaced with a mixed aqueous solution of sodium dihydrogen phosphate and glucose, and all other operations and conditions were the same as in Example 1.

[0266] The mixed aqueous solution is prepared as follows: 0.68g sodium dihydrogen phosphate, 0.06g sodium hydroxide, and 4.7g glucose are diluted with water to 100ml. The pH of the system is 6.5.

[0267] The drug-liposome ratio and LPC content of tracer liposome solutions prepared from ultrafiltrates of different secondary ultrafiltration processes are shown in the table below.

[0268] Table 18 Effects of different tracer liposomes

[0269] In the tracer liposome solutions prepared in the embodiments of the present invention, the tracer content is approximately 10 mg / mL.

[0270] The animal experiment procedure (Figures 1-5) was as follows: SD rats were weighed and anesthetized by intraperitoneal injection of 5% chloral hydrate (0.7 mL / 100 g). The skin of the rat's hind thigh root was prepared, and the drug was injected subcutaneously into the sole of the hind limb. 20 μL of tracer was injected (injection completed in 50-60 seconds). The sole of the foot was pressed for 3 minutes after injection. 30 minutes after drug administration, the thigh skin was cut open, and the stained popliteal lymph nodes were found in the muscle crevices of the rat's leg (Figures 1-4). The same method was used to inject into the forelimb, and the axillary lymph nodes were found (Figure 5).

[0271] Figure 1 shows the tracer images of rat lymph nodes in liposome solutions with different particle sizes. In Figure 1, the particle sizes in parts A to C are 70 nm (Example 59), 200 nm (Example 60), and 131 nm (Example 1), respectively. As shown in Figure 1, severe staining occurs with a particle size of 70 nm, while sentinel lymph nodes show full staining with particle sizes of 131 nm and 200 nm.

[0272] In actual use, the liposome solutions obtained in Examples 61-62 showed similar characteristics to those in Examples 1 and 60, with no smudging and full staining of sentinel lymph nodes.

[0273] Figure 2 shows rat tracing images using different encapsulating agents. Part A of Figure 2 shows encapsulation with ammonium sulfate in Example 1, and Part B shows encapsulation with sucrose octasulfate in Example 62. As can be seen from Figure 2, ammonium sulfate encapsulation resulted in clearer staining of liposomes.

[0274] Figures 3 and 4 show a comparison of rat lymph node staining after encapsulation with different staining agents using the same process. In Figure 3, part A shows the mitoxantrone liposome solution prepared in Example 63; parts B of Figure 3 and Part B of Figure 4 show the methylene blue liposome solution prepared in Example 1; and part A of Figure 4 shows the indocyanine green liposome solution prepared in Example 64. As can be seen from the figures, using the preparation method of this invention, methylene blue, mitoxantrone, and indocyanine green can all achieve encapsulation and in vivo lymph node tracing.

[0275] Figure 5 shows a comparison of rat lymph node tracing using commercially available methylene blue injection and the methylene blue liposome solution from Example 1. In the figure, 1 represents commercially available methylene blue injection, and 2 represents the methylene blue liposome solution from Example 1. As can be seen from the figure, the secondary lymph nodes were not stained by the methylene blue liposome solution from Example 1.

[0276] Figure 6. Animal experiment procedure: After weighing the SD rats, they were anesthetized by intraperitoneal injection of 5% chloral hydrate (injection dose was 0.7 mL / 100 g). The skin of the rat's abdomen was prepared, and 20 μL of tracer was injected subcutaneously into the abdomen (injection was completed in 50-60 seconds). The rats were observed 24 hours later.

[0277] Figure 6 compares the subcutaneous irritation of commercially available methylene blue injection, mitoxantrone hydrochloride injection, and methylene blue liposome solution from Example 1 in rats. In the figures, 1 represents commercially available methylene blue injection, 2 represents methylene blue liposome solution from Example 1, and 3 represents commercially available mitoxantrone hydrochloride injection. Figure 6 illustrates that the methylene blue liposome solution from Example 1 exhibits less local irritation.

[0278] Figure 7 Animal experiment procedure: After weighing, SD rats were anesthetized by intraperitoneal injection of 5% chloral hydrate (injection dose was 0.7 mL / 100 g), and 20 μL of tracer was injected subcutaneously into the sole of the forelimb (injection completed in 50-60 seconds). Observation was performed 24 hours later.

[0279] Figure 7 shows a comparison of the irritation differences between the methylene blue liposome solution of Example 1 and the commercially available nano-carbon suspension injection. In the figure, 1 represents the commercially available nano-carbon suspension injection, and 2 represents the methylene blue liposome solution of Example 1. As shown in Figure 7, the methylene blue liposome solution of Example 1 causes less local irritation.

[0280] The animal experiments described above demonstrate that the liposome solution of the present invention is far less irritating than currently available lymph node tracers when used for lymph node tracing.

[0281] Figure 8 shows photographs of the free drug in the product after encapsulation with methylene blue and ammonium sulfate at different molar ratios (without secondary ultrafiltration) passing through a gel column (the product was passed through a gel column during the intermediate encapsulation efficiency test). In Figure 8, the molar ratios of methylene blue to ammonium sulfate in sections A through C are 2.5:1 (Example 17), 2.0:1 (Example 1), and 1.5:1 (Example 16), respectively. As shown in Figure 8, different molar ratios of methylene blue and ammonium sulfate can effectively encapsulate methylene blue, resulting in less free drug.

Claims

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

2. The tracer liposome solution as described in claim 1, characterized in that, The tracer liposome solution satisfies one or more of the following conditions: (1) The phospholipids include distearate phosphatidylcholine and / or hydrogenated soybean phospholipids; (2) The mass ratio of the phospholipid to the cholesterol is (1-10):1, preferably (2-9):1, for example 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 is ≥ (1:1), preferably ≥ (1.5:1), for example 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 70nm, preferably 70nm-200nm, more preferably 80nm-200nm, such as 90nm, 100nm, 131nm, 132nm, 133nm, 134nm, 150nm or 180nm.

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

4. The tracer liposome solution as described in claim 1, characterized in that, The tracer liposome solution satisfies one or more of the following conditions: (1) The drug-to-lipid ratio is ≥42%, preferably 42%-81%; for example, 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) LPC content <0.76mg / mL, such as 0.02mg / mL, 0.03mg / mL, 0.04mg / mL, 0.05mg / mL, 0.06mg / mL, 0.07mg / mL, 0.08mg / mL, 0.09mg / mL, 0.10mg / mL, 0.11mg / mL, 0.12mg / mL, 0.14 mg / mL, 0.15mg / mL, 0.16mg / mL, 0.18mg / mL, 0.21mg / mL, 0.26mg / mL, 0.28mg / mL, 0.29mg / mL, 0.37mg / mL, 0.40mg / mL, 0.48mg / mL, 0.5mg / mL, 0.57mg / mL or 0.76mg / mL; (3) The total impurity content of related substances is 0.91%, 0.92% or 0.95%; (4) The tracer concentration is ≥4 mg / mL, preferably ≥9 mg / mL, for example 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 as described in any one of claims 1-4, characterized in that, It includes the following steps: S1. A phospholipid solution was injected into an internal aqueous phase solution, aged, extruded, and ultrafiltered to obtain a blank liposome solution. The phospholipid solution comprises phospholipids, cholesterol, and a solvent; the phospholipids include one or more of distearate, hydrogenated soybean phospholipids, and sphingomyelin. S2. The blank liposome solution is added to the tracer solution, mixed and encapsulated, and then ultrafiltered. The encapsulation time is not less than 5 minutes, and the pH value of the system during encapsulation 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, the pH value of the system during encapsulation 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, for example 68°C; (2) In S2, the encapsulation time is 10-55 min, for example 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, or 50 min; and, (3) In S2, during the encapsulation, the pH value of the system 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 method for preparing the phospholipid solution includes: dissolving phospholipids and cholesterol in a solvent, heating, and stirring to dissolve; In S1, the phospholipid solution contains bisstearoylphosphatidylcholine and / or hydrogenated soybean phospholipids; In S1, the concentration of phospholipid in the phospholipid solution is 237-711 mg / mL, preferably 400-800 mg / mL, for example 423 mg / mL, 474 mg / mL, 500 mg / mL, 708 mg / mL or 750 mg / mL; In S1, the mass ratio of phospholipids to cholesterol in the phospholipid solution is (1-10):1, preferably (2-9):1, for example 3:1, 4:1, 5:1 or 7:1; In S1, the volume ratio of the phospholipid solution to the internal aqueous phase solution is 1:(2-20), preferably 1:5, 1:10 or 1:15; In S1, the method for preparing the internal aqueous solution includes the following steps: dissolving the encapsulating agent in water, heating, and stirring to dissolve; In S1, the encapsulating agent in the internal aqueous phase solution includes ammonium sulfate and / or sucrose octasulfate, preferably ammonium sulfate; In S1, the concentration of the encapsulating agent in the internal aqueous phase solution is 0.2-1M, preferably 0.2-0.6M, for example 0.4M or 0.5M; In S1, when the phospholipid solution is injected into the internal aqueous phase solution, the temperature of both solutions is not lower than 60°C, for example, 63°C, 68°C or 73°C. In S1, the ripening temperature is 63℃-73℃, for example, 63℃, 68℃ or 73℃; In S1, during the ripening process, the rotation speed is 150-250 rpm, for example, 200 rpm; In S1, the extrusion operation includes: first extruding through a polycarbonate film with a pore size of 200 nm, and then extruding through a polycarbonate film with a pore size of 80 nm. In S1, the ultrafiltration can be performed using a 300KD ultrafiltration membrane for constant volume concentration ultrafiltration; and, In S1, the number of ultrafiltration cycles is ≥8.

9. 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 actual detection concentration of the encapsulating agent in the blank liposome solution is 0.04-0.09M, for example 0.05M, 0.0625M or 0.0833M; The actual detected concentration of the encapsulating agent refers to the concentration of the remaining encapsulating agent in the blank liposome solution after ultrafiltration to remove the unencapsulated encapsulating agent in step S1. (2) In S2, the tracer solution contains one or more of methylene blue, indocyanine green and mitoxantrone, such as methylene blue, indocyanine green or mitoxantrone, preferably methylene blue; (3) In S2, the tracer solution contains a tracer concentration of no more than 3% by mass, for example, 1.48%; (4) In S2, the molar ratio of the tracer in the tracer solution to the encapsulating agent actually detected in the blank liposome solution is ≥ (1:1), preferably ≥ (1.5:1), for example 2.0:1, 2.5:1 or 3:1; (5) In S2, the ultrafiltrate of the ultrafiltration is a mixed aqueous solution of glucose and HEPES, a mixed aqueous solution of sodium dihydrogen phosphate and glucose, or a mixed aqueous solution of sodium chloride and HEPES. In the glucose and HEPES mixed aqueous solution, the concentration of HEPES is preferably 3.5-5.5 mg / mL, for example 4.05 mg / mL; and the concentration of glucose is preferably 40-50 mg / mL, for example 47 mg / mL. In the aqueous solution of sodium dihydrogen phosphate and glucose, the concentration of sodium dihydrogen phosphate is preferably 6-8 mg / mL, for example 6.8 mg / mL; and the concentration of glucose is preferably 40-50 mg / mL, for example 47 mg / mL. In the mixed aqueous solution of sodium chloride and HEPES, the sodium chloride concentration is preferably 5-12 mg / mL, for example 8.45 mg / mL; the HEPES concentration is preferably 3-5 mg / mL, for example 4.1 mg / mL. (9) In S2, the pH value of the ultrafiltrate is 2-11, preferably 3-10, for example 4, 6.5, 7, 8 or 9; (6) In S2, the ultrafiltration is performed using a 300KD ultrafiltration membrane for constant volume concentration ultrafiltration; and, (7) In S2, the number of ultrafiltrations is ≥10 times.

10. A tracer liposome solution prepared by any one of claims 5-9.

11. A method for preparing a tracer liposome solution, characterized in that, It includes the following steps: S1. A phospholipid solution was injected into an internal aqueous phase solution, aged, extruded, and ultrafiltered to obtain a blank liposome solution. The phospholipid solution comprises phospholipids, cholesterol, and a solvent; the phospholipids include one or more of distearate, hydrogenated soybean phospholipids, and sphingomyelin; and the maturation time is at least 5 minutes. S2. The blank liposome solution is added to the tracer solution, mixed and encapsulated, and then ultrafiltered.

12. The preparation method according to claim 11, characterized in that, It meets one or more of the following conditions: (1) In S1, the concentration of phospholipid in the phospholipid solution is 237-711 mg / mL; (2) In S1, the mass ratio of phospholipid to cholesterol in the phospholipid solution is (1-10):1; (3) In S1, the volume ratio of the phospholipid solution to the internal aqueous phase solution is 1:(2-20); (4) In S1, the encapsulating agent in the internal aqueous phase solution includes ammonium sulfate and / or sucrose octasulfate; (5) In S1, the concentration of the encapsulating agent in the internal aqueous phase solution is 0.2-1M; (6) In S1, the ripening time is 5 min-2 h; and, (7) In S1, the ripening temperature is 63℃-73℃.

13. The preparation method according to claim 11, characterized in that, It meets one or more of the following conditions: (1) In S1, the concentration of phospholipid in the phospholipid solution is 400-800 mg / mL; (2) In S1, the mass ratio of phospholipid to cholesterol in the phospholipid solution is (2-9):1; (3) In S1, the volume ratio of the phospholipid solution to the internal aqueous phase solution is 1:5, 1:10 or 1:15; (4) In S1, the encapsulating agent in the internal aqueous phase solution is ammonium sulfate; (5) In S1, the concentration of the encapsulating agent in the internal aqueous phase solution is 0.2-0.6M; (6) In S1, the ripening time is 15-60 min; and, (7) In S1, the ripening temperature is 63°C, 68°C or 73°C.

14. The preparation method according to claim 11, characterized in that, It meets one or more of the following conditions: (1) In S1, the concentration of phospholipid in the phospholipid solution is 423 mg / mL, 474 mg / mL, 500 mg / mL, 708 mg / mL or 750 mg / mL; (2) In S1, the mass ratio of phospholipid to cholesterol in the phospholipid solution is 3:1, 4:1, 5:1 or 7:1; (3) In S1, the concentration of the encapsulating agent in the internal aqueous phase solution is 0.4M or 0.5M; and, (4) In S1, the ripening time is 30 min.

15. The preparation method according to claim 11, characterized in that, It meets one or more of the following conditions: (1) In S1, the method for preparing the phospholipid solution includes the following steps: dissolving phospholipids and cholesterol in a solvent, heating, and stirring to dissolve; (2) In S1, the phospholipid solution contains bisstearoylphosphatidylcholine and / or hydrogenated soybean phospholipids; (3) In S1, the solvent in the phospholipid solution is an alcohol solvent, such as anhydrous ethanol; (4) In S1, the method for preparing the internal aqueous solution includes the following steps: dissolving the encapsulating agent in water, heating, and stirring to dissolve; (5) In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the temperature of both solutions is not lower than 60°C, for example, 63°C, 68°C or 73°C; (6) In S1, when the phospholipid solution is injected into the inner aqueous phase solution, the injection time is 5-10 min; (7) In S1, during the ripening process, the rotation speed is 150-250 rpm, for example 200 rpm; (8) In S1, the extrusion operation includes: first extruding through a polycarbonate film with a pore size of 200 nm, and then extruding through a polycarbonate film with a pore size of 80 nm. (9) In S1, after the extrusion and before the ultrafiltration, the system is cooled to ≤25℃; (10) In S1, the ultrafiltration is performed using a 300KD ultrafiltration membrane for constant volume concentration ultrafiltration; and, (11) In S1, the number of ultrafiltrations is ≥ 8 times.

16. The preparation method according to claim 11, characterized in that, It meets one or more of the following conditions: (1) In S2, the actual detection concentration of the encapsulating agent in the blank liposome solution is 0.04-0.09M; the actual detection concentration of the encapsulating agent refers to the concentration of the remaining encapsulating agent in the blank liposome solution after ultrafiltration to remove the unencapsulated encapsulating agent in step S1. (2) In S2, the tracer solution contains one or more of methylene blue, indocyanine green, and mitoxantrone; (3) In S2, the molar ratio of the tracer in the tracer solution to the encapsulating agent actually detected in the blank liposome solution is ≥ (1:1); the encapsulating agent actually detected refers to the encapsulating agent remaining in the blank liposome solution after the unencapsulated encapsulating agent is removed by ultrafiltration in step S1. (4) In S2, the encapsulation temperature is not lower than 55°C; (5) In S2, the encapsulation time shall not be less than 5 minutes; (6) In S2, the system pH value is 6-11 during encapsulation; and, (7) In S2, the pH value of the ultrafiltrate is 2-11.

17. The preparation method according to claim 16, characterized in that, It meets one or more of the following conditions: (1) In S2, the actual detection concentration of the encapsulating agent in the blank liposome solution is 0.05M, 0.0625M or 0.0833M; (2) In S2, the tracer solution contains methylene blue, indocyanine green, or mitoxantrone; (3) In S2, the molar ratio of the tracer in the tracer solution to the encapsulating agent actually detected in the blank liposome solution is ≥ (1.5:1); (4) In S2, the encapsulation temperature is 60-76℃; (5) In S2, the encapsulation time is 5 min-60 min; (6) In S2, during the encapsulation process, the pH of the system is 6.5-10.5; and, (7) In S2, the pH value of the ultrafiltrate is 3-10.

18. The preparation method according to claim 17, characterized in that, It meets one or more of the following conditions: (1) In S2, the tracer in the tracer solution is methylene blue; (2) In S2, the molar ratio of the tracer in the tracer solution to the encapsulating agent actually detected in the blank liposome solution is 2.0:1, 2.5:1 or 3:1; (3) In S2, the encapsulation temperature is 65-70°C, for example 68°C; (4) In S2, the encapsulation time is 10-55 min, for example 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min or 50 min; (5) In S2, during the encapsulation process, the pH value of the system is 7, 7.5, 8, 8.5, 9, 9.8, 10, 10.5, or 11; and, (6) In S2, the pH value of the ultrafiltrate is 4, 6.5, 7, 8 or 9.

19. The preparation method according to claim 11, characterized in that, It meets one or more of the following conditions: (1) In S2, the method for preparing the tracer solution includes the following steps: mixing the tracer and the solvent, and heating to dissolve; the preferred temperature for heating to dissolve is 35-45°C, for example, 40°C; (2) In S2, when the blank liposome solution is added to the tracer solution, the temperature of the tracer solution is 30-40°C, for example, 35°C; (3) In S2, the solvent in the tracer solution is water for injection; (4) In S2, the tracer solution contains a tracer concentration of no more than 3% by mass, for example, 1.48%; (5) In S2, after encapsulation, the temperature is reduced to ≤25℃; (6) In S2, the ultrafiltrate of the ultrafiltration is a mixed aqueous solution of glucose and HEPES, a mixed aqueous solution of sodium dihydrogen phosphate and glucose, or a mixed aqueous solution of sodium chloride and HEPES. In the glucose and HEPES mixed aqueous solution, the concentration of HEPES is preferably 6-8 mg / mL, for example 4.05 mg / mL; the concentration of glucose is preferably 40-50 mg / mL, for example 47 mg / mL. In the aqueous solution of sodium dihydrogen phosphate and glucose, the concentration of sodium dihydrogen phosphate is preferably 6-8 mg / mL, for example 6.8 mg / mL; and the concentration of glucose is preferably 40-50 mg / mL, for example 47 mg / mL. In the mixed aqueous solution of sodium chloride and HEPES, the sodium chloride concentration is preferably 5-12 mg / mL, for example 8.45 mg / mL; the HEPES concentration is preferably 3-5 mg / mL, for example 4.1 mg / mL. (7) In S2, the ultrafiltration is performed using a 300KD ultrafiltration membrane for constant volume concentration ultrafiltration; and, (8) In S2, the number of ultrafiltrations is ≥10.

20. A tracer liposome solution prepared by any one of claims 11-19.

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