Preparation process for research and development of small-scale test and pilot-scale test system of various multi-vesicular liposomes

Abstract

The invention belongs to the technical field of multi-vesicular liposome preparation, and particularly relates to a preparation process for researching and developing a small-scale test and pilot-scale test system of multiple multi-vesicular liposomes, which comprises the following specific steps: S1, respectively feeding an organic phase and a water phase into an organic phase tank; starting a shearing machine at the bottom of the organic phase tank to emulsify the organic phase and the water phase, so that the organic phase, the water phase and the shearing machine are self-circulated to be fully dispersed and emulsified to form an inner water phase; s2, adding a certain amount of water phase into the aeration tank; s3, transferring the emulsified inner water phase in the organic phase tank to an aeration tank, and then continuously introducing nitrogen into the aeration tank; the method has the characteristics of extremely good homogeneity effect and good stability of the multi-vesicular liposome at present, and a part of emulsion with relatively uniform size can be obtained without further centrifugation or screening by a screen, so that the method is relatively convenient to use in small-scale test and pilot-scale test preparation systems.

Description

Technical Field

[0001] The invention relates to the technical field of multivesicular liposome preparation, in particular to a preparation process for developing small-scale and pilot-scale systems of various multivesicular liposomes. Background Art

[0002] The word "liposome" appears more often in beauty-related products, giving people a sense of cutting-edge and high-end. Phospholipids can spontaneously form bilayer vesicles in water, which are named liposomes. Liposomes are self-assembled bodies of lipid molecules or lipids. Liposomes are bilayer structures spontaneously formed by lipid molecules or lipids in water.

[0003] Since the basic structure of the plasma membrane of organisms is also a phospholipid bilayer membrane, liposomes have a structure similar to that of biological cells and therefore have good biocompatibility, so they are also called "artificial biomembranes" and "artificial cells". Liposomes use this compatibility to achieve targeted or efficient drug delivery. Ideal liposomes should have good tissue compatibility, low toxicity, and appropriate drug encapsulation and release capabilities.

[0004] The structure of liposomes is also diverse. Large-sized liposomes with multiple cavities are called multivesicular liposomes, which play an important role in the sustained and controlled release of drugs, while temperature-sensitive thermosensitive liposomes can selectively release or exert their effects on drugs in different parts due to temperature differences.

[0005] At present, it is difficult to accurately control the particle size of most multivesicular liposomes. This also leads to the insufficient application of multivesicular liposomes in vaccine adjuvants, molecular diagnosis, etc. If it can have small size controllability and selectivity based on the advantages of loading and release, it will greatly meet the new needs of pharmaceutical preparations and lead a series of important research directions.

[0006] The traditional preparation process of the double emulsion method is to first prepare colostrum by homogenization, then prepare water-in-oil-in-water double emulsion by homogenization, and then remove the excess oil phase by nitrogen or rotary evaporation. This method currently has the characteristics of poor uniformity and stability of multivesicular liposomes, and further centrifugation or sieve screening is required to obtain a part of the emulsion with relatively uniform size. Therefore, in the preparation system of small and pilot tests, it is necessary to systematically take into account multiple preparation methods for the convenience of research. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation process for developing a variety of multivesicular liposomes in small and pilot systems, so as to solve the problem that the particle size of most multivesicular liposomes currently mentioned in the above background technology is difficult to accurately control. This also leads to the problem that multivesicular liposomes are insufficiently applied in vaccine adjuvants, molecular diagnosis, etc.

[0008] To achieve the above object, the present invention provides the following technical solution: a preparation process for developing a variety of multivesicular liposome small-scale and pilot-scale systems, and the specific steps of the preparation process for developing a variety of multivesicular liposome small-scale and pilot-scale systems are as follows:

[0009] S1: The organic phase and the aqueous phase are respectively put into the organic phase tank, and the shearing machine at the bottom of the organic phase tank is started to emulsify the organic phase and the aqueous phase, so that the organic phase, the aqueous phase and the shearing machine are self-circulated and fully dispersed and emulsified to form an inner aqueous phase;

[0010] S2: Add a certain amount of water phase into the aeration tank;

[0011] S3: The emulsified inner water phase in the organic phase tank is transferred to the aeration tank, and then nitrogen is continuously introduced into the aeration tank;

[0012] The aeration tank is started to stir, the temperature control is started, and the volatile organic phase in the solution is removed. The volatile organic phase passes through a condenser, and the volatile organic phase is condensed and recovered into a waste liquid tank;

[0013] S4; connecting the final material in the aeration tank to the ultrafiltration system to remove small molecules and unencapsulated inner water phase, and quantitatively replenishing the water phase to the aeration tank to set the volume to form the final small-scale test and pilot-scale products;

[0014] S5: Discharge the waste liquid from the ultrafiltration process of the ultrafiltration system in step S4 into a waste liquid tank.

[0015] Preferably, an oil phase tank and a tubular shearing machine are arranged at the organic phase tank, the oil phase tank is connected with the organic phase tank through the tubular shearing machine, the oil phase is stored in the oil phase tank, and in the step S1, during the emulsification operation, the valve corresponding to the oil phase tank and the switch of the tubular shearing machine are opened, and the oil phase in the oil phase tank is contacted and mixed with the organic phase and the aqueous phase through the shearing action of the tubular shearing machine and the shearing machine.

[0016] Preferably, in step S1, during the emulsification operation, the shearing machine at the bottom of the organic phase tank and the tubular shearing machine are both variable frequency controllable shearing machines to control the line speed and the processing volume.

[0017] Preferably, a weighing module and an online conductivity meter are arranged inside the organic phase tank.

[0018] Preferably, a weighing module, a bottom stirring mechanism and an online conductivity meter are provided on the lower side of the aeration tank.

[0019] Preferably, the organic phase is chloroform or methanol.

[0020] Preferably, in step S1, the organic phase and the aqueous phase are outputted through plunger pump 1 and plunger pump 2 respectively, and the organic phase and the aqueous phase are mixed through a pipeline mixer by variable frequency quantitative adjustment of plunger pump 1 and plunger pump 2, and the mixed organic phase and aqueous phase are emulsified.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This solution will adjust the organic phase and the aqueous phase by frequency conversion and quantitative adjustment, mix them through a specific pipeline mixer, and enter the tubular shearing machine for emulsification. Through the setting of the shearing machine, the organic phase, the aqueous phase and the oil phase are fully mixed. To form an emulsified inner water phase, nitrogen is continuously introduced into the aeration tank to start stirring to treat the inner water phase, start temperature control, and remove most of the volatile organic phase (such as chloroform, methanol, etc.) in the solution. The waste gas passes through the condenser and is condensed and recycled into the waste liquid tank. The final material in the aeration tank is connected to the ultrafiltration system to remove small molecules and unencapsulated inner water phases, and the aeration tank can be quantitatively supplemented with water phase and constant volume to form the final small-scale and pilot-scale products.

[0023] This method currently has the characteristics of excellent uniformity and stability of multivesicular liposomes. It does not require further centrifugation or sieve screening to obtain a portion of emulsion with relatively uniform size. Therefore, it is relatively convenient to use in small-scale and pilot-scale preparation systems. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Embodiment 1:

[0026] A preparation process for developing a variety of multivesicular liposome small-scale and pilot-scale systems, characterized in that the specific steps of the preparation process for developing a variety of multivesicular liposome small-scale and pilot-scale systems are as follows:

[0027] S1: The organic phase and the aqueous phase are respectively put into the organic phase tank, and the shearing machine at the bottom of the organic phase tank is started to emulsify the organic phase and the aqueous phase, so that the organic phase, the aqueous phase and the shearing machine are self-circulated and fully dispersed and emulsified to form an inner aqueous phase; the organic phase is chloroform, methanol, etc.;

[0028] An oil phase tank and a tubular shearing machine are arranged at the organic phase tank. The oil phase tank is connected with the organic phase tank through the tubular shearing machine. The oil phase is stored in the oil phase tank. During the emulsification operation, the valve corresponding to the oil phase tank and the switch of the tubular shearing machine are opened. The oil phase in the oil phase tank is contacted and mixed with the organic phase and the water phase through the shearing action of the tubular shearing machine and the shearing machine.

[0029] During the emulsification operation, the shearing machine at the bottom of the organic phase tank and the tubular shearing machine are both variable frequency controllable shearing machines to control the line speed and processing volume;

[0030] A weighing module and an online conductivity meter are arranged inside the organic phase tank.

[0031] S2: Add a certain amount of water phase into the aeration tank; a weighing module, a bottom stirring mechanism, and an online conductivity meter 2 are arranged on the lower side of the aeration tank.

[0032] S3: The emulsified inner water phase in the organic phase tank is transferred to the aeration tank, and then nitrogen is continuously introduced into the aeration tank;

[0033] The aeration tank is started to stir, the temperature control is started, and the volatile organic phase in the solution is removed. The volatile organic phase passes through a condenser, and the volatile organic phase is condensed and recovered into a waste liquid tank;

[0034] S4; connecting the final material in the aeration tank to the ultrafiltration system to remove small molecules and unencapsulated inner water phase, and quantitatively replenishing the water phase to the aeration tank to set the volume to form the final small-scale test and pilot-scale products;

[0035] S5: Discharge the waste liquid from the ultrafiltration process of the ultrafiltration system in step S4 into a waste liquid tank.

[0036] The prepared multivesicular liposomes have excellent uniformity and good stability. No further centrifugation or sieve screening is required to obtain a portion of emulsion with relatively uniform size. Therefore, it is convenient to use in small-scale and pilot-scale preparation systems.

[0037] Embodiment 2:

[0038] A preparation process for developing a variety of multivesicular liposome small-scale and pilot-scale systems, characterized in that the specific steps of the preparation process for developing a variety of multivesicular liposome small-scale and pilot-scale systems are as follows:

[0039] S1: The organic phase and the aqueous phase are respectively put into the organic phase tank, and the shearing machine at the bottom of the organic phase tank is started to emulsify the organic phase and the aqueous phase, so that the organic phase, the aqueous phase and the shearing machine are self-circulated and fully dispersed and emulsified to form an inner aqueous phase; the organic phase is chloroform, methanol, etc.;

[0040] The organic phase and the aqueous phase are outputted through plunger pump 1 and plunger pump 2 respectively. By variable frequency quantitative adjustment of plunger pump 1 and plunger pump 2, the organic phase and the aqueous phase are mixed through a pipeline mixer. The mixed organic phase and aqueous phase are emulsified and then enter the organic phase tank.

[0041] During the emulsification operation, the shearing machine at the bottom of the organic phase tank and the tubular shearing machine are both variable frequency controllable shearing machines to control the line speed and processing volume;

[0042] A weighing module and an online conductivity meter are arranged inside the organic phase tank.

[0043] S2: Add a certain amount of water phase into the aeration tank; a weighing module, a bottom stirring mechanism, and an online conductivity meter 2 are arranged on the lower side of the aeration tank.

[0044] Online Conductivity meter It is a new generation of all-Chinese microcomputer-based high-end instrument, with all-Chinese display, Chinese menu-based operation, full intelligence, multi-function, high measurement performance, strong environmental adaptability, etc. The secondary meter can measure the conductivity of general liquids when equipped with electrodes with constants of 1.0 or 10; it can accurately measure the conductivity of pure water or ultrapure water when equipped with electrodes with constants of 0.1 or 0.01. Conductivity , especially suitable for online continuous monitoring of the conductivity of high-purity water such as boiler feed water and steam condensate in power plants.

[0045] The Chinese line conductivity meter simplifies the functions while ensuring the performance, thus having a particularly strong price advantage. Strong environmental adaptability, clear display, simple operation and excellent test performance make it very cost-effective. It can be widely used in thermal power, chemical fertilizer, metallurgy, environmental protection, pharmaceutical, biochemistry, food and Tap water Continuous monitoring of conductivity values ​​in solutions such as

[0046] S3: The emulsified inner water phase in the organic phase tank is transferred to the aeration tank, and then nitrogen is continuously introduced into the aeration tank;

[0047] Nitrogen is a single substance formed by the nitrogen element, with the chemical formula N2. It is a colorless and odorless gas at room temperature and pressure. It can only react with hydrogen to form ammonia under high temperature, high pressure and catalyst conditions. It can also react with oxygen to form nitric oxide under discharge. Even active metals such as Ca, Mg, Sr and Ba can only react with it under heating.

[0048] The high chemical stability of nitrogen is related to its molecular structure. Two N atoms are combined by a triple bond to form a nitrogen molecule, which contains one σ bond and two π bonds. Because the π bond is the first to be attacked in a chemical reaction, and the energy level of the π bond in the N2 molecule is lower than that of the σ bond, it is difficult to open the π bond, making it difficult for N2 to participate in chemical reactions.

[0049] Nitrogen is relatively stable. As the driving gas for aeration and mixing of the internal water phase, it is not easy to react as a whole, thus ensuring the quality of the product.

[0050] The aeration tank is started to stir, the temperature control is started, and the volatile organic phase in the solution is removed. The volatile organic phase passes through a condenser, and the volatile organic phase is condensed and recovered into a waste liquid tank;

[0051] S4; connecting the final material in the aeration tank to the ultrafiltration system to remove small molecules and unencapsulated inner water phase, and quantitatively replenishing the water phase to the aeration tank to set the volume to form the final small-scale test and pilot-scale products;

[0052] S5: Discharge the waste liquid from the ultrafiltration process of the ultrafiltration system in step S4 into a waste liquid tank.

[0053] The prepared multivesicular liposomes have excellent uniformity and good stability. No further centrifugation or sieve screening is required to obtain a portion of emulsion with relatively uniform size. Therefore, it is convenient to use in small-scale and pilot-scale preparation systems.

[0054] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description, and it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation process for developing a variety of multivesicular liposomes in small and pilot test systems, characterized in that: The specific steps of the preparation process for developing various multivesicular liposome small-scale and pilot-scale systems are as follows: S1: The organic phase and the aqueous phase are respectively put into the organic phase tank, and the shearing machine at the bottom of the organic phase tank is started to emulsify the organic phase and the aqueous phase, so that the organic phase, the aqueous phase and the shearing machine are self-circulated and fully dispersed and emulsified to form an inner aqueous phase; S2: Add a certain amount of water phase into the aeration tank; S3: The emulsified inner water phase in the organic phase tank is transferred to the aeration tank, and then nitrogen is continuously introduced into the aeration tank; The aeration tank is started to stir, the temperature control is started, and the volatile organic phase in the solution is removed. The volatile organic phase passes through a condenser, and the volatile organic phase is condensed and recovered into a waste liquid tank; S4; connecting the final material in the aeration tank to the ultrafiltration system to remove small molecules and unencapsulated inner water phase, and quantitatively replenishing the water phase to the aeration tank to set the volume to form the final small-scale test and pilot-scale products; S5: Discharge the waste liquid in the ultrafiltration process of the ultrafiltration system in step S4 into a waste liquid tank.

2. A preparation process for developing a variety of multivesicular liposomes in small and pilot test systems according to claim 1, characterized in that: An oil phase tank and a tubular shearing machine are arranged at the organic phase tank. The oil phase tank is connected with the organic phase tank through the tubular shearing machine. The oil phase is stored in the oil phase tank. In the step S1, during the emulsification operation, the valve corresponding to the oil phase tank and the switch of the tubular shearing machine are opened. The oil phase in the oil phase tank is contacted and mixed with the organic phase and the aqueous phase through the shearing action of the tubular shearing machine and the shearing machine.

3. A preparation process for developing a variety of multivesicular liposomes in small and pilot test systems according to claim 2, characterized in that: In the step S1, during the emulsification operation, the shearing machine at the bottom of the organic phase tank and the tubular shearing machine are both variable frequency controllable shearing machines to control the line speed and the processing volume.

4. A preparation process for developing a variety of multivesicular liposomes in small and pilot test systems according to claim 1, characterized in that: A weighing module and an online conductivity meter are arranged inside the organic phase tank.

5. A preparation process for developing a variety of multivesicular liposomes in small and pilot test systems according to claim 1, characterized in that: A weighing module, a bottom stirring mechanism and an online conductivity meter 2 are arranged on the lower side of the aeration tank.

6. A preparation process for developing a variety of multivesicular liposomes in small and pilot test systems according to claim 1, characterized in that: The organic phase is chloroform or methanol.

7. The preparation process for developing a variety of multivesicular liposomes in small and pilot test systems according to claim 1, characterized in that: In step S1, the organic phase and the aqueous phase are outputted through plunger pump 1 and plunger pump 2 respectively, and the organic phase and the aqueous phase are mixed through a pipeline mixer by variable frequency quantitative adjustment of plunger pump 1 and plunger pump 2, and the mixed organic phase and aqueous phase are emulsified.