CN112120022A

CN112120022A - Blank multivesicular liposome and preparation method and device thereof

Info

Publication number: CN112120022A
Application number: CN202011044785.4A
Authority: CN
Inventors: 王磊; 曹雄飞; 秦敦忠
Original assignee: Jiangsu Sinvo Chemical Technology Co ltd
Current assignee: Jiangsu Sinvo Chemical Technology Co ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2020-12-25

Abstract

The invention discloses a blank multivesicular liposome and a preparation method and a device thereof, belonging to the field of pesticide auxiliary agents. The preparation raw materials of the multivesicular liposome comprise phospholipid substances, sterol substances, neutral liposome, fatty acid ester substances and vitamins, the multivesicular liposome containing high electronegativity and steric hindrance vesicles can be obtained, and when the multivesicular liposome is used as a pesticide auxiliary agent, the coagulation of the multivesicular liposome can be effectively inhibited, the drug release time is prolonged, and the drug effect is improved; meanwhile, the invention provides a method and a device for preparing blank multi-vesicular liposome, wherein a continuous flow microchannel reactor is used for replacing the traditional high-speed shearing equipment.

Description

Blank multivesicular liposome and preparation method and device thereof

Technical Field

The invention belongs to the field of pesticide auxiliary agents, and particularly relates to a blank multivesicular liposome and a preparation method and a device thereof.

Background

Multivesicular liposomes (MVL) are composed of a plurality of non-concentric aqueous drug solution vesicles separated by continuous non-concentric lipid bilayer, and have high encapsulation efficiency for water-soluble drugs. In particular, multivesicular liposomes release drug through ruptured vesicles, while intact vesicles remain intact. The multivesicular liposome is a non-concentric circle topological structure, so that a reservoir is formed at an injection part, the medicament encapsulated in the multivesicular liposome is gradually released along with the continuous degradation of a lipid bilayer to generate a good slow release effect, the burst release effect is avoided, and the effect of controlling the medicament release time from several days to several weeks can be obtained by adjusting a preparation formula and process parameters. The multivesicular liposome can also well solve the problems of small encapsulated volume of the common liposome, low drug-loading rate, immediate release of the drug once the lipid membrane is broken and the like.

The preparation method of the multi-capsule system is more, and due to the structural particularity of the multi-capsule system, the multiple emulsion method is a main reported method for preparing MVL at present, and comprises the following specific steps of firstly dissolving a plurality of lipid components in a volatile organic solvent to form an oil phase, wherein the organic solvent can be a mixed solvent of dichloromethane, chloroform, diethyl ether and the like, the used lipid generally comprises a negative-charge phospholipid amphoteric phospholipid neutral lipid (common triacylglycerol) and cholesterol, selecting a proper oil-water volume ratio to mix a drug-containing aqueous solution (an internal aqueous phase) with the oil phase, preparing a water-in-oil colostrum (W/O) type colostrum at room temperature by methods of ultrasound, high-speed shear dispersion, vortex mixing, nozzle atomization and the like, then rapidly adding the colostrum into an isotonic external aqueous phase with a certain volume, and carrying out vortex or mechanical shear to emulsify again under a certain condition to form the W/O/W type multiple emulsion (the oil chamber contains a plurality of aqueous chambers In the phase, the organic solvent is removed at a suitable temperature, the resulting mixture is concentrated by centrifugation and freed of the drug, the outer aqueous phase is replaced by a suitable storage and physiologically acceptable salt solution (e.g., 0.9% NaCl solution), and finally the drug content is adjusted and filled to obtain MVL.

The general production process of the multivesicular liposome is divided into four continuous unit operations, specifically, the formation of primary emulsion, the formation of multiple emulsion, the removal of organic solvent and microfiltration. Wherein the formation of the multiple emulsion and the removal of the organic solvent are carried out in the same reaction tank. In each unit operation, there are many conditions to be controlled, such as mixing speed, time, diameter of stirring paddle, etc. in the emulsification process, or gas flow rate, bubble rate, temperature, etc. in the solvent removal process, and filtration rate, circulation rate, buffer exchange volume, etc. in the microfiltration process, which can affect particle size, particle size distribution, yield, etc., and many factors need to be precisely controlled, otherwise, the quality of the final product is affected. The preparation of multivesicular liposome generally adopts the high-speed shearing method, when the high-speed shearing multivesicular liposome colostrum (10000-18000r/min), can locally produce high temperature, the temperature of production can be greater than the phase transition temperature of multivesicular liposome and lead to multivesicular liposome structure inseparable, reveal, particle diameter grow scheduling problem, because traditional shearing cauldron space is big, the shear plane is certain, the mass transfer effect is not good, production efficiency is low, the liposome particle diameter size that obtains is uneven need to handle once more a great deal of problems such as just can use simultaneously. And the vesicle has too large particle size and is difficult to penetrate a target wax layer, an air hole and the like, so the method is not suitable for application in the field of pesticide preparations.

Therefore, how to obtain the environment-friendly multivesicular liposome which has good stability of vesicle, easy biodegradation, stable property, uniform and moderate particle size distribution and can wrap the pesticide active substance and be applied to the pesticide field becomes a technical problem to be solved in the field.

Disclosure of Invention

1. Problems to be solved

Aiming at the problems of the existing preparation method that the obtained multivesicular liposome has an untight structure, poor stability of the vesicle, overlarge and uneven particle size and the like, the invention provides a preparation method of a blank multivesicular liposome, and the continuous flow microchannel reactor is utilized to obtain the environment-friendly multivesicular liposome with good stability of the vesicle and even particle size distribution;

aiming at the problems of incompact structure, leakage, overlarge and uneven particle size and the like of the multi-vesicular liposome caused by the fact that traditional high-speed shearing equipment is adopted in the existing multi-vesicular liposome preparation process, the invention provides a blank multi-vesicular liposome preparation device, wherein a continuous flow microchannel reactor is used for replacing the traditional high-speed shearing equipment, so that the stability and uniformity of the vesica of the multi-vesicular liposome are improved, and the size of the vesica is reduced;

aiming at the problems that the prior multivesicular liposome has uneven vesicle particle size, needs to be processed again before use, is difficult to permeate a target wax layer, air holes and the like, and is not suitable for the field of pesticide preparations, the invention provides the environment-friendly multivesicular liposome which has good vesicle stability, uniform and moderate particle size distribution and can wrap pesticide active substances and be applied to the field of pesticides.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

a blank multivesicular liposome, wherein the D50 particle size range of the multivesicular liposome is 1-6 μm, and the Span is 1.01-1.07.

Preferably, the preparation raw material of the multivesicular liposome comprises the following components in parts by weight

40-80 parts of phospholipid substances;

10-40 parts of sterol substances;

5-10 parts of neutral liposome;

1-5 parts of fatty acid ester substances;

0.01-0.05 parts of vitamin.

Preferably, the phospholipid is phospholipid and/or hydrogenated derivatives of phospholipid, specifically one or more selected from the group consisting of soybean phospholipid, phosphatidic acid, egg yolk phospholipid, sunflower phospholipid, rapeseed phospholipid, canola phospholipid, linseed phospholipid, castor oil phospholipid, and hydrogenated derivatives of the above phospholipids.

Preferably, the sterol substance is sterol and/or sterol derivative, and the sterol includes one or more selected from beta-sitosterol, beta-sitostanol, stigmasterol, stigmastanol, campesterol, campestanol, ergosterol, avenasterol, brassicasterol and cholesterol; the sterol derivative comprises one or more of sterol sulfonate, sterol sulfate, sterol phosphate, sterol alcohol ether sulfonate, sterol alcohol ether sulfate and sterol alcohol ether phosphate. (ii) a The sterol alcohol ether and sterol alcohol ether salt polyoxyethylene ether thereof have the molar molecular weight of 44-3200, and preferably 1000-2400.

Preferably, the neutral liposome is one or more selected from the group consisting of triolein, tricaprylin, tributyrin, tripalmitin, trimyristin, trilinolein, trihexanoic acid and tricaprin acid.

Preferably, the fatty acid ester material is one or more selected from trimethylolpropane fatty acid ester, propylene glycol fatty acid ester, pentaerythritol fatty acid ester, polyethylene glycol fatty acid monoester, polyethylene glycol fatty acid diester, fatty acid polyoxyethylene ether fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid, sucrose fatty acid ester or the material with the general formula (2);

in the general formula (2), R is C1-C57 straight chain or branched chain alkyl, unsaturated hydrocarbon chain, polyglycerol residue, sucrose residue, sorbitol residue or sorbitan residue; r₁，R₂，R₃H, CH are represented independently of each other₃Or CH₂CH₃；n₁，n₂，n₃Independently represent an integer of 0 to 30, and n is an integer of 1 to 8; z is C1-C18 straight chain alkyl or unsaturated hydrocarbon chain;

preferably, the vitamin is one or two of vitamin E and vitamin C.

Preferably, the multivesicular liposome further comprises preparation auxiliary materials, wherein the preparation auxiliary materials comprise sucrose, glucose, L-lysine and cason.

Preferably, the multivesicular liposome also comprises a preparation solvent, wherein the preparation solvent is a mixed solvent of diethyl ether and chloroform, and the volume ratio of the diethyl ether to the chloroform is (0.8-1.5): 1.

A preparation device of blank multivesicular liposome can be used for preparing the blank multivesicular liposome, and comprises a feeding unit, a micro-reaction unit communicated with the feeding unit, a collection kettle communicated with the micro-reaction unit, and a solvent removal unit communicated with the collection kettle; a back pressure valve is arranged on a communication pipeline between the micro-reaction unit and the collection kettle;

the micro-reaction unit comprises a plurality of micro-reaction groups which are connected in series, each micro-reaction group comprises a continuous flow micro-channel reactor (hereinafter referred to as a micro-channel reactor), a micro-mixer and a delay tube which are sequentially communicated, wherein the delay tube of the former micro-reaction group is communicated with the micro-channel reactor of the latter micro-reaction group;

the feeding unit comprises a treatment kettle, a metering pump, a check valve and a constant temperature pipe which are sequentially communicated, and the feeding unit is communicated with the micro-channel reactor of the micro-reaction unit through the constant temperature pipe;

the solvent removing unit comprises a finished product kettle communicated with the collecting kettle, a condenser communicated with the finished product kettle, a solvent collecting tank and a vacuum pump communicated with the condenser, and a tail gas treatment device communicated with the vacuum pump; meanwhile, the finished product kettle is also provided with a nitrogen inlet.

Preferably, a water bath heating device is further included for water bath heating of the micro reaction group.

Preferably, n microreaction groups, where n is a natural number and 2. ltoreq. n.ltoreq.10.

Preferably, the microchannel reactor is provided with a plurality of microchannels, the equivalent diameter of the section of each microchannel is 0.01-10 mm, and the length of each microchannel is 0.2-2 m; the micro mixer is provided with a plurality of micro channels, the equivalent diameter of the cross section of each micro channel is 0.01-10 mm, and the length of each micro channel is 0.4-8.5 m. Particularly, the fluid A enters a feed inlet of the microchannel reactor after passing through a constant temperature tube of 0.1-5 m, and the diameter of the constant temperature tube is 1-5 mm; the diameter of the channel of the delay tube is 1-5 mm, and the length of the channel is 0.5-15 m.

A method for preparing blank multivesicular liposomes, comprising the steps of:

1) preparing an oil phase as a fluid A, an inner water phase as a fluid B and an outer water phase as a fluid C;

2) alternately mixing the fluid A, B by using a continuous flow microchannel reactor and a micro mixer until colostrums W/O with the particle size D50 of 1-6 mu m and Span of 1.01-1.07 are prepared, wherein the colostrums W/O is used as a fluid D;

3) and mixing the fluid C, D by using a continuous flow microchannel reactor and a micromixer in sequence to prepare a multiple emulsion W/O/W with the D50 of 1-6 mu m and the Span of 1.01-1.07, wherein the multiple emulsion W/O/W is used as a fluid E.

4) And (3) carrying out solvent removal treatment on the fluid E to obtain the multivesicular liposome.

Preferably, in the step 1), phospholipid substances, sterol substances, neutral liposomes, vitamins and fatty acid ester substances are dissolved in a solvent to prepare the fluid A, wherein the mass fraction of the solvent is 75-90%;

the fluid B is a sucrose and/or glucose aqueous solution with the concentration of 4-7 wt%;

the fluid C is an aqueous solution containing L-lysine, glucose and carbazone, wherein the concentration of the L-lysine is 30-50 mmol/L, the concentration of the glucose is 4-7 wt%, and the concentration of the carbazone is 0.01-0.06 wt%.

Preferably, in the step 2), the treatment temperature of the fluid is 0-38 ℃, preferably 5-30 ℃, and more preferably 10-15 ℃.

Preferably, in the step 2), the collision pressure P when the fluid A and the fluid B are mixed is 1-30 MPa; and the flow rate ratio of the fluid A to the fluid B is 1 (0.1-1).

Preferably, in the step 2), the residence time t1 of the fluid in the micro reaction unit is 60-900 s.

Preferably, in the step 3), the total residence time t2 of the fluid in the micro reaction group is 10-15 s; wherein the flow rate ratio of the fluid D to the fluid C is 1 (1-10).

Preferably, the preparation of multivesicular liposomes using the above-mentioned apparatus for preparing multivesicular liposomes having n microreaction groups is as follows:

1) preparing an oil phase, an inner water phase and an outer water phase by using different treatment kettles respectively, and sequentially naming the two phases as a fluid A, a fluid B and a fluid C;

2) introducing the fluid A, B into a first micro-reaction group, and mixing in a micro-channel reactor to generate primary emulsion W/O as a fluid D; then, continuously introducing the fluid D into the micro mixer, and then continuously introducing the next n-2 micro reaction groups for mixing until the particle size D50 of the fluid D is 1-6 mu m and the Span is 1.01-1.07; during the process, the working pressure and the flow rate of the fluid A, B in the system are controlled by the mutual matching of a metering pump, a back pressure valve, the number of micro-reaction groups, the length and the section equivalent diameter of a micro-channel in the micro-reaction group, the length and the section equivalent diameter of a delay pipe and the section equivalent diameter, and finally the control of the collision pressure P (the collision pressure P is 1-30MPa, preferably 26MPa) when the fluid A, B is mixed and the total residence time t1 (the t1 is 60-900 s) of the fluid D in the micro-reaction unit are realized;

3) continuously introducing the fluid D and the fluid C into the next micro-reaction group for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; similarly, the flow rate of the fluid D, C and the total residence time t2(t2 is 10-15 s) of the fluid E in the micro-reaction unit are controlled by the mutual matching of a metering pump, a back pressure valve, the length and the section equivalent diameter of the micro-channel in the micro-reaction group, the length and the section equivalent diameter of the delay tube;

4) introducing the fluid E into a collection kettle for collection under the condition of heat preservation; then, introducing the fluid E collected in the collection kettle into a finished product kettle, simultaneously introducing nitrogen for solvent removal, under the action of a condenser, removing the solvent, storing the diluted finished product in a solvent collection tank, and detecting to obtain the multivesicular liposome;

5) diluting with 0.9% NaCl or PBS (pH 7.5-8.2) buffer solution to obtain the multivesicular liposome preparation.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) the multivesicular liposome provided by the invention has the advantages of compact structure, good stability of vesicles, small particle size (the range of the particle size D50 is 1-6 mu m), and uniform particle size;

(2) the multivesicular liposome provided by the invention can be spontaneously assembled and wrap active substances in water. When in use, the multi-vesicular liposome can be directly added into a diluted spray preparation, has better wrapping capacity on hydrophilic and lipophilic medicaments, can effectively fuse wax layers on the surfaces of target crops, improves the absorption and conduction of the medicaments on the targets or the target plants, achieves the aim of reducing application and improving efficiency, and can replace or reduce the application of other types of synergists in the field of pesticides;

(3) according to the multivesicular liposome provided by the invention, sterol derivatives are used for replacing the traditional sterol, so that the electronegativity and steric hindrance of vesicles are improved, the coagulation of the multivesicular liposome is effectively inhibited, the drug release time of the multivesicular liposome is prolonged, and the drug effect is improved;

(4) according to the preparation device of the multivesicular liposome, the continuous flow microchannel reactor is used for carrying out the configuration of colostrum and re-emulsification, so that the traditional high-speed shearing equipment is abandoned, and the problems that the structure of the multivesicular liposome is not compact, the leakage is caused and the particle size is increased due to the fact that the high temperature is locally generated by high-speed shearing in the process of preparing the colostrum of the multivesicular liposome by the traditional high-speed shearing equipment and the generated temperature is higher than the phase transition temperature of the multivesicular liposome are solved; the mass transfer effect is poor due to a certain shearing surface caused by an overlarge shearing space, the production efficiency is low, the obtained liposome has uneven particle size and can be used only by being treated again, and the like;

the continuous flow microchannel reactor has great advantages in replacing the traditional high-speed shearing equipment, and the main reasons are that the piston diameter of the power part of the microchannel reactor equipment is small, the stroke is long, the output homogeneity is high pressure duration and stable pressure, and the inner diameter of a pore channel is very small, so the energy conversion rate is high, the pulse fluctuation is very small, the process conditions of sample particles passing through the microfluidic interaction cavity are basically the same, and the homogeneous sample Span is close to 1 and can directly meet the requirements;

the multivesicular liposome prepared by the continuous flow microchannel reactor has uniform particle size, moderate size and smooth edge, and the Span is close to 1.01-1.07, so that the problem of the application of the traditional high-speed shearing equipment can be effectively solved;

(5) the preparation device of the multivesicular liposome provided by the invention has the advantages that the process is continuous flow reaction, the reaction time is shortened to several minutes, and the reaction efficiency and the production efficiency are obviously improved. The microchannel reactor has almost no amplification effect, has higher safety performance and is suitable for industrial production;

(6) according to the preparation method of the multivesicular liposome, the control of fluid collision pressure during the preparation of primary emulsion is realized by adjusting the back pressure valve; meanwhile, the micro-reaction time during the preparation of the primary emulsion W/O is realized by regulating and controlling the number of micro-reaction groups, the length of the delay tube and the flow rate of the fluid; and the removal rate of the solvent, the temperature of the whole system and the like are added, so that the control of the vesicle particle size and the Span distribution is realized.

Drawings

FIG. 1 is a schematic structural view of an apparatus for preparing multivesicular liposomes according to an embodiment of the present invention;

in the figure: 100. a feed unit; 110. an oil phase feed group; 111. a treatment kettle A; 112. a metering pump A; 113. a check valve A; 114. a constant temperature tube A; 120. an internal aqueous phase feed group; 121. a treatment kettle B; 122. a metering pump B; 123. a check valve B; 124. a constant temperature tube B; 130. an external aqueous phase feed group; 131. a treatment kettle C; 132. a metering pump C; 133. a check valve C; 134. a thermostatic tube C;

200. a micro-reaction unit; 210. a micro-reaction group; 211. a microchannel reactor; 212. a micro mixer; 213. a delay tube;

300. a back pressure valve;

400. collecting the kettle;

500. a pressure gauge;

600. a solvent removal device; 610. a finished product kettle; 620. a condenser; 630. a solvent collection tank; 640. a vacuum pump; 650. and a tail gas collecting device.

Detailed Description

As shown in fig. 1, the preparation apparatus of multivesicular liposomes provided by the present invention comprises a feeding unit 100, a micro-reaction unit 200 communicated with the feeding unit 100, a water bath heating device for heating the micro-reaction unit 210, a collection vessel 400 communicated with the micro-reaction unit 200, and a desolvation unit 600 communicated with the collection vessel 400; a back pressure valve 300 is also arranged on a communication pipeline between the micro-reaction unit 200 and the collection kettle 400; the micro-reaction unit 200 comprises a plurality of micro-reaction groups 210 which are connected in series, wherein each micro-reaction group 210 comprises a micro-channel reactor 211, a micro-mixer 212 and a delay tube 213 which are sequentially communicated, and the delay tube 213 of the former micro-reaction group 210 is communicated with the micro-channel reactor 211 of the latter micro-reaction group 210; the feeding unit 100 comprises a treatment kettle, a metering pump, a check valve and a thermostatic tube which are sequentially communicated, and the feeding unit 100 is communicated with the micro-channel reactor 211 of the micro-reaction unit 200 through the thermostatic tube.

The desolventizing unit 600 comprises a finished product kettle 610 communicated with the collecting kettle 400 through a pipeline (provided with a switch valve), a condenser 620 communicated with the finished product kettle 610 through a pipeline, a solvent collecting tank 630 and a vacuum pump 640 which are respectively communicated with the condenser 620 through pipelines, and a tail gas treatment device 650 communicated with the vacuum pump 640 through a pipeline; meanwhile, the finished product kettle 610 is also provided with a nitrogen inlet.

As shown in fig. 1, the feed unit 100 includes an oil phase feed set 110, an inner aqueous phase feed set 120, and an outer aqueous phase feed set 130; the oil phase feeding group 110 is composed of a treatment kettle A111, a metering pump A112, a check valve A113 and a thermostatic tube 114 which are connected in series in sequence through pipelines; similarly, the inner aqueous phase feeding group 120 is composed of a treatment kettle B121, a metering pump B122, a check valve B123 and a constant temperature pipe B124 which are connected in series in sequence through pipelines; the external aqueous phase feed group 130 is composed of a treatment tank C131, a metering pump C132, a check valve C133, and a thermostatic tube C134, which are connected in series in this order via pipes.

As shown in FIG. 1, the micro-reaction unit 200 may be formed by connecting 2-20 micro-reaction groups 210 in series according to practical situations; wherein the microchannel reactor 211 is provided with a plurality of microchannels, the equivalent diameter of the section of each microchannel is 0.01-10 mm, and the length of each microchannel is 0.2-2 m; the micro mixer is provided with a plurality of micro channels, the equivalent diameter of the cross section of each micro channel is 0.01-10 mm, and the length of each micro channel is 0.4-8.5 m. Particularly, the fluid A enters a feed inlet of the microchannel reactor after passing through a constant temperature tube of 0.1-5 m, and the diameter of the constant temperature tube is 1-5 mm; the diameter of the channel of the delay tube is 1-5 mm, and the length of the channel is 0.5-15 m.

The technical solutions of the present invention are further clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1. Wherein, the processing kettle A111 and the processing kettle B121 are both 2L reaction kettles, and the processing kettle C131 is a 15L conventional pilot scale reaction kettle; the number n of the micro-reaction groups 210 is 5; wherein the length of each thermostatic tube (A114, B124 and C134) is 0.3m, and the diameter of each thermostatic tube is 3 mm; the diameter of the delay tube 213 is 3mm, and the length is 1 m; the equivalent diameter of the section of the micro-channel reactor 211 is 0.5mm, and the length of the micro-channel is 1 m; the micromixer 212 has a plurality of microchannels having a cross-sectional equivalent diameter of 0.3mm and a microchannel length of 0.5 m.

In this example, the preparation method of the multivesicular liposome specifically comprises the following steps:

1) preparation of oil phase fluid a: adding 80g of soybean phospholipid, 40g of cholesterol, 10g of triolein, 5g of tween 80 and 0.0325g of vitamin C into 1244.25g of chloroform diethyl ether solution (V: V is 1:1) in advance into a treatment kettle A111, stirring for dissolving, and keeping the temperature at 0-38 ℃ to obtain fluid A for later use;

preparation of inner aqueous phase fluid B: adding a 7 wt% glucose aqueous solution into a treatment kettle B121 in total of 1382.5g in advance, and keeping the temperature at 0-38 ℃ to serve as a fluid B for later use;

preparation of external aqueous phase fluid C: PBS buffer solution with pH of 7.5, L-lysine aqueous solution, glucose aqueous solution and cason aqueous solution are added into a treatment kettle C131 in advance, 5400g of mixed solution with L-lysine concentration of 50mmol/L, glucose concentration of 4 wt% and cason concentration of 0.02 wt% is obtained by mixing finally, and the mixed solution is named as fluid C for standby after heat preservation at 0-38 ℃.

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212, then enters the delay tube 213, and then continues to enter the next 3 micro reaction groups 210 for mixing; during the period, the temperature of the micro reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro reaction unit 200 was adjusted to be maintained between 1 and 30.0MPa by a back pressure valve 300; finally, the residence time of the colostrum W/O fluid D in the micro-reaction unit 200 was set to 300s, and the grain size D10 of the colostrum W/O fluid D was 0.57 μm, D50 was 5.1 μm, D90 was 5.87 μm, and Span was 1.04 by sampling.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:1.95 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreactor unit 200 was maintained at 1-30MPa by the back pressure valve 300, the residence time of the multiple emulsion W/O/W fluid E was 15s by the adjustment of the metering pumps in conjunction with the length of the delay tube 213, the particle size D10 of the multiple emulsion W/O/W fluid E was 0.53 μm, D50 was 5.5 μm, D90 was 6.36 μm, and Span was 1.06.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, introducing the fluid E collected in the collection kettle 400 into a finished product kettle 610, simultaneously introducing nitrogen for solvent removal, under the action of a condenser 620, removing the solvent, and storing the diluted finished product in a solvent collection tank 630 to obtain the polycystic liposome with the solid content of 2% after detection, wherein the particle size D10 is 0.58, the particle size D50 is 5.7 microns, the particle size D90 is 6.68 microns, and the particle size Span is 1.07, named 1A; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Example 2

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1. Wherein, the processing kettle A111 and the processing kettle B121 are both 2L reaction kettles, and the processing kettle C131 is a 15L conventional pilot scale reaction kettle; the number n of the micro-reaction groups 210 is 6; wherein the length of each thermostatic tube (A114, B124 and C134) is 0.2m, and the diameter thereof is 4 mm; the diameter of the delay tube 213 is 2mm, and the length is 6 m; the equivalent diameter of the section of the micro-channel reactor 211 is 3mm, and the length of the micro-channel is 2 m; the micromixer 212 has a plurality of microchannels having a cross-sectional equivalent diameter of 4mm and a microchannel length of 0.8 m.

1) preparation of oil phase fluid a: adding yolk phospholipid 40g, cholesterol 10g, tricaprylin 5g, tween 80 1g and vitamin C0.01042 g into processing kettle A111, dissolving in chloroform ether solution 170g (V: V is 0.8:1), stirring for dissolving, and keeping the temperature at 0-38 deg.C to obtain fluid A;

preparation of inner aqueous phase fluid B: adding a 4 wt% glucose aqueous solution into a treatment kettle B121 in advance, wherein 266g of the aqueous solution is kept at the temperature of 0-38 ℃ for later use as a fluid B;

preparation of external aqueous phase fluid C: adding PBS buffer solution with pH of 8.2, L-lysine aqueous solution, glucose aqueous solution and cason aqueous solution into a treatment kettle C131 in advance, finally mixing to obtain 2562g of mixed solution with L-lysine concentration of 30mmol/L, glucose concentration of 7 wt% and cason concentration of 0.02 wt% serving as an external water phase, and preserving heat at 0-38 ℃ to name fluid C for later use.

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212, then enters the delay tube 213, and then continues to enter the next 4 micro reaction groups 210 for mixing; during the period, the temperature of the micro reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro reaction unit 200 was adjusted to be maintained between 1 and 30.0MPa by a back pressure valve 300; finally, the residence time of the colostrum W/O fluid D was set to 500s, and the grain size D10, D50, D90 and Span of the colostrum W/O fluid D were measured to be 0.43 μm, 4.6 μm, 5.17 μm and 1.03, respectively.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:4.81 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreactor unit 200 was maintained at 1-30MPa by the back pressure valve 300, the residence time of the multiple emulsion W/O/W fluid E was 10s by the adjustment of the metering pumps in conjunction with the length of the delay tube 213, the particle size D10 of the multiple emulsion W/O/W fluid E was 0.41 μm, the particle size D50 was 4.4 μm, the particle size D90 was 4.98 μm, and the Span was 1.04.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, introducing the fluid E collected in the collection kettle 400 into a finished product kettle 610, simultaneously introducing nitrogen for solvent removal, under the action of a condenser 620, removing the solvent, and storing the diluted finished product in a solvent collection tank 630 to obtain the polycystic liposome with the solid content of 2% after detection, wherein the particle size D10 is 0.47 mu m, the particle size D50 is 4.7 mu m, the particle size D90 is 5.5 mu m, and the particle size Span is 1.05, named 2A; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Example 3

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1. Wherein, the processing kettle A111 and the processing kettle B121 are both 2L reaction kettles, and the processing kettle C131 is a 15L conventional pilot scale reaction kettle; the number n of the micro-reaction groups 210 is 3; wherein the length of each thermostatic tube (A114, B124 and C134) is 0.11m, and the diameter thereof is 4 mm; the diameter of the delay tube 213 is 4mm, and the length is 0.7 m; the equivalent diameter of the section of the micro-channel reactor 211 is 9mm, and the length of the micro-channel is 1.9 m; the micromixer 212 has a plurality of microchannels having a cross-sectional equivalent diameter of 8mm and a microchannel length of 7 m.

1) preparation of oil phase fluid a: adding 50g of soybean phospholipid, 20g of cholesterol, 15g of tributyrin, 5g of tween 60 and 0.045g of vitamin C into a treatment kettle A111 in advance, dissolving in 535g of chloroform ether solution (V: V is 0.9:1), stirring for dissolving, and keeping the temperature at 0-38 ℃ to obtain a fluid A for later use;

preparation of inner aqueous phase fluid B: adding a 4 wt% glucose aqueous solution into a treatment kettle B121 in advance, wherein the total amount of the solution is 630g, and keeping the temperature at 0-38 ℃ to be used as a fluid B for later use;

preparation of external aqueous phase fluid C: adding PBS buffer solution with pH of 7.8, L-lysine aqueous solution, glucose aqueous solution and cason aqueous solution into a treatment kettle C131 in advance, finally mixing to obtain 2520g of mixed solution with L-lysine concentration of 40mmol/L, glucose concentration of 5 wt% and cason concentration of 0.05 wt% as external water phase, and preserving heat at 0-38 ℃ to name fluid C for later use.

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212 and then enters the delay tube 213, and then continues to enter the next micro reaction group 1 210 for mixing; during the period, the temperature of the micro reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro reaction unit 200 was adjusted to be maintained between 1 and 30.0MPa by a back pressure valve 300; finally, the residence time of the colostrum W/O fluid D in the micro-reaction unit 200 was set to 600s, and the grain size D10 of the colostrum W/O fluid D was 0.41 μm, D50 was 4.1 μm, D90 was 4.6 μm, and Span was 1.02 by sampling.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:2 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreaction unit 200 was maintained at 1-30MPa by the back pressure valve 300, the particle size D10 of the W/O/W fluid E was 0.39 μm, D50 was 4.2 μm, D90 was 4.63 μm, and Span was 1.01 by adjustment of the metering pumps and the length of the delay tube 213 were matched.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, introducing the fluid E collected in the collection kettle 400 into a finished product kettle 610, simultaneously introducing nitrogen for solvent removal, under the action of a condenser 620, removing the solvent, and storing the diluted finished product in a solvent collection tank 630 to obtain the multivesicular liposome with the solid content of 3% after detection, wherein the particle size D10 is 0.44 μm, the particle size D50 is 4.5 μm, the particle size D90 is 5.0 μm, and the particle size Span is 1.01, named 3A; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Example 4

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1. Wherein, the processing kettle A111 and the processing kettle B121 are both 2L reaction kettles, and the processing kettle C131 is a 15L conventional pilot scale reaction kettle; the number n of the micro-reaction groups 210 is 8; wherein the length of each thermostatic tube (A114, B124 and C134) is 0.4m, and the diameter thereof is 4.5 mm; the diameter of the delay tube 213 is 4.5mm, and the length is 1 m; the equivalent diameter of the section of the micro-channel reactor 211 is 0.5mm, and the length of the micro-channel is 0.6 m; the micromixer 212 has a plurality of microchannels having a cross-sectional equivalent diameter of 3mm and a microchannel length of 0.7 m.

1) preparation of oil phase fluid a: adding 70g of soybean phospholipid, 40g of cholesterol, 15g of triolein, 2g of T80 and 0.0254g of vitamin C into 1165g of chloroform diethyl ether solution (V: V is 1:1) in advance in a treatment kettle A111, stirring for dissolving, and keeping the temperature at 0-38 ℃ to obtain fluid A for later use;

preparation of inner aqueous phase fluid B: adding 130g of 4 wt% glucose aqueous solution into a treatment kettle B121 in advance, and keeping the temperature at 0-38 ℃ to serve as fluid B for later use;

preparation of external aqueous phase fluid C: PBS buffer solution with the pH value of 7.8, L-lysine aqueous solution, glucose aqueous solution and cason aqueous solution are added into a treatment kettle C131 in advance, 8506g of mixed solution with the L-lysine concentration of 40mmol/L, the glucose concentration of 5 wt% and the cason concentration of 0.03 wt% is finally obtained by mixing, and the mixed solution is named as fluid C for standby after heat preservation at the temperature of 0-38 ℃.

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212, then enters the delay tube 213, and then continues to enter the next 6 micro reaction groups 210 for mixing; during the period, the temperature of the micro reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro reaction unit 200 was adjusted to be maintained between 1 and 30.0MPa by a back pressure valve 300; finally, the residence time of the colostrum W/O fluid D is 400s, the grain size D10 of the colostrum W/O fluid D is 0.54 μm, D50 is 5.2 μm, D90 is 5.95 μm, and Span is 1.04 by sampling detection.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:6 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreactor unit 200 was maintained at 1-30MPa by the back pressure valve 300, the residence time of the multiple emulsion W/O/W fluid E was 15s by the adjustment of the metering pumps in conjunction with the length of the delay tube 213, the particle diameter D10 of the multiple emulsion W/O/W fluid E was 0.53 μm, D50 was 5.0 μm, D90 was 5.68 μm, and Span was 1.03.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, the fluid E collected in the collection kettle 400 is introduced into a finished product kettle 610, nitrogen is introduced for solvent removal, under the action of a condenser 620, the solvent is removed, the diluted finished product enters a solvent collection tank 630 for storage, and the polycystic liposome with the solid content of 2% is obtained after detection, wherein the particle size D10 is 0.58 μm, the particle size D50 is 5.6 μm, the particle size D90 is 6.57 μm, and the particle size Span is 1.07, named 4A; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Example 5

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1. Wherein, the processing kettle A111 and the processing kettle B121 are both 2L reaction kettles, and the processing kettle C131 is a 15L conventional pilot scale reaction kettle; the number n of the micro-reaction groups 210 is 5; wherein the length of each thermostatic tube (A114, B124 and C134) is 4m, and the diameter of each thermostatic tube is 3 mm; the diameter of the delay tube 213 is 4mm, and the length is 3 m; the equivalent diameter of the section of the micro-channel reactor 211 is 8mm, and the length of the micro-channel is 1.5 m; the micromixer 212 has a plurality of microchannels having a cross-sectional equivalent diameter of 7mm and a microchannel length of 3 m.

1) preparation of oil phase fluid a: adding 80g of yolk phospholipid and soybean phospholipid, 30g of cholesterol, 15g of tributyrin, 5g of Tween 80 and 0.026g of vitamin C into 1073g of chloroform diethyl ether solution (V: V is 1:1) in advance, stirring for dissolving, and keeping the temperature at 0-38 ℃ to obtain fluid A for later use;

preparation of inner aqueous phase fluid B: adding a 5 percent by weight glucose aqueous solution into a treatment kettle B121 in advance, wherein 1205g of the aqueous solution is kept at the temperature of 0-38 ℃ for later use as a fluid B;

preparation of external aqueous phase fluid C: PBS buffer solution with pH of 7.8, L-lysine aqueous solution, glucose aqueous solution and cason aqueous solution are added into a treatment kettle C131 in advance, 5425g of mixed solution with L-lysine concentration of 40mmol/L, glucose concentration of 5 wt% and cason concentration of 0.02 wt% is obtained by mixing finally, and the mixed solution is named as fluid C for standby after heat preservation at 0-38 ℃.

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212, then enters the delay tube 213, and then continues to enter the next 3 micro reaction groups 210 for mixing; during the period, the temperature of the micro reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro reaction unit 200 was adjusted to be maintained between 1 and 30.0MPa by a back pressure valve 300; finally, the residence time of the colostrum W/O fluid D was set to 200s, and the grain size D10, D50, D90 and Span of the colostrum W/O fluid D were measured to be 0.29 μm, 3.4 μm, 3.83 μm and 1.04, respectively.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:4.81 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreactor unit 200 was maintained at 1-30MPa by the back pressure valve 300, the residence time of the multiple emulsion W/O/W fluid E was 15s by the adjustment of the metering pumps in conjunction with the length of the delay tube 213, the particle size D10 of the multiple emulsion W/O/W fluid E was 0.34 μm, the particle size D50 was 3.3 μm, the particle size D90 was 3.73 μm, and the Span was 1.03.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, introducing the fluid E collected in the collection kettle 400 into a finished product kettle 610, simultaneously introducing nitrogen for solvent removal, under the action of a condenser 620, removing the solvent, and storing the diluted finished product in a solvent collection tank 630 to obtain the polycystic liposome with the solid content of 2% after detection, wherein the particle size D10 is 0.37 μm, the particle size D50 is 3.5 μm, the particle size D90 is 4.05 μm, and the particle size Span is 1.05, named 5A; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Example 6

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1. Wherein, the processing kettle A111 and the processing kettle B121 are both 2L reaction kettles, and the processing kettle C131 is a 15L conventional pilot scale reaction kettle; the number n of the micro-reaction groups 210 is 20; wherein the length of each thermostatic tube (A114, B124 and C134) is 0.3m, and the diameter thereof is 4 mm; the diameter of the delay tube 213 is 4mm, and the length is 2 m; the equivalent diameter of the section of the micro-channel reactor 211 is 9mm, and the length of the micro-channel is 1.9 m; the micromixer 212 has a plurality of microchannels having a cross-sectional equivalent diameter of 8mm and a microchannel length of 7.6 m.

1) preparation of oil phase fluid a: adding 80g of castor oil phospholipid and soybean phospholipid, 30g of cholesterol, 15g of tricaprylin, 5g of tween 20 and 0.027g of vitamin C into 1073g of chloroform diethyl ether solution (V: V is 1:1) in advance, stirring for dissolving, and keeping the temperature at 0-38 ℃ to obtain fluid A for later use;

preparation of external aqueous phase fluid C: PBS buffer solution with pH of 7.8, L-lysine aqueous solution, glucose aqueous solution and cason aqueous solution are added into a treatment kettle C131 in advance, 5425g of mixed solution with L-lysine concentration of 30mmol/L, glucose concentration of 4 wt% and cason concentration of 0.025 wt% is obtained by mixing finally, and the mixed solution is named as fluid C for standby after heat preservation at 0-38 ℃.

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212 and then enters the delay tube 213, and then continues to enter the next 18 micro reaction groups 210 for mixing; during the period, the temperature of the micro reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro reaction unit 200 was adjusted to be maintained between 1 and 30.0MPa by a back pressure valve 300; finally, the residence time of the colostrum W/O fluid D in the micro-reaction unit 200 was set to 200s, and the grain size D10 of the colostrum W/O fluid D was 0.48 μm, D50 was 4.2 μm, D90 was 4.93 μm, and Span was 1.06 by sampling.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:4.5 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreaction unit 200 was maintained at 1-30MPa by the back pressure valve 300, and by the adjustment of the metering pumps and the length of the delay tube 213 being matched for 15s, the W/O/W fluid E particles D10 was sampled and detected to be 0.44 μm, D50 was 4.1 μm, D90 was 4.74 μm, and Span was 1.05.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, the fluid E collected in the collection kettle 400 is introduced into a finished product kettle 610, nitrogen is introduced for solvent removal, under the action of a condenser 620, the solvent is removed, the diluted finished product enters a solvent collection tank 630 for storage, and the multivesicular liposome with the solid content of 2% is obtained after detection, wherein the particle size D10 is 0.52 μm, D50 is 4.6 μm, D90 is 5.79 μm, Span is 1.07, and the name is 6A.

Example 7

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1. Wherein, the processing kettle A111 and the processing kettle B121 are both 2L reaction kettles, and the processing kettle C131 is a 15L conventional pilot scale reaction kettle; the number n of the micro-reaction groups 210 is 10; wherein the length of each thermostatic tube (A114, B124 and C134) is 0.98m, and the diameter thereof is 2 mm; the diameter of the delay tube 213 is 4mm, and the length is 14 m; the equivalent diameter of the section of the micro-channel reactor 211 is 9mm, and the length of the micro-channel is 1.98 m; the micromixer 212 has a plurality of microchannels with a cross-sectional equivalent diameter of 0.04mm and a microchannel length of 8.3 m.

1) preparation of oil phase fluid a: adding 80g of yolk phospholipid, 40g of cholesterol and cholesterol sulfate, 15g of triolein, 1g of Tween 85 and 0.0290g of vitamin C into 1017g of chloroform diethyl ether solution (V: V is 1.2:1) in advance in a treatment kettle A111, stirring for dissolving, and keeping the temperature at 0-38 ℃ to obtain fluid A for later use;

preparation of inner aqueous phase fluid B: adding 4 wt% glucose aqueous solution into the treatment kettle B121 in total 1150g, and keeping the temperature at 0-38 ℃ to serve as fluid B for later use;

preparation of external aqueous phase fluid C: PBS buffer solution with pH of 7.8, L-lysine aqueous solution, glucose aqueous solution and cason aqueous solution are added into a treatment kettle C131 in advance, 8098g of mixed solution with L-lysine concentration of 40mmol/L, glucose concentration of 5 wt% and cason concentration of 0.05 wt% is finally obtained by mixing, and fluid C is named after heat preservation at 0-38 ℃ for standby.

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212, then enters the delay tube 213, and then continues to enter the next 8 micro reaction groups 210 for mixing; during the period, the temperature of the micro reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro reaction unit 200 was adjusted to be maintained between 1 and 30.0MPa by a back pressure valve 300; finally, the residence time of the colostrum W/O fluid D is 500s, and the grain size D10 of the colostrum W/O fluid D is 0.21 μm, D50 is 1.3 μm, D90 is 1.58 μm and Span is 1.06 by sampling detection.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:3.51 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreactor unit 200 was maintained at 1-30MPa by the back pressure valve 300, the residence time of the multiple emulsion W/O/W fluid E was 15s by the adjustment of the metering pumps in conjunction with the length of the delay tube 213, the particle size D10 of the multiple emulsion W/O/W fluid E was 0.19 μm, the particle size D50 was 1.2 μm, the particle size D90 was 1.45 μm, and the Span was 1.05.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, introducing the fluid E collected in the collection kettle 400 into a finished product kettle 610, simultaneously introducing nitrogen for solvent removal, under the action of a condenser 620, removing the solvent, and storing the diluted finished product in a solvent collection tank 630 to obtain the multivesicular liposome with the solid content of 2% after detection, wherein the particle size D10 is 0.23 μm, the particle size D50 is 1.5 μm, the particle size D90 is 1.83 μm, and the particle size Span is 1.07, named 7A; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Example 8

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1. Wherein, the processing kettle A111 and the processing kettle B121 are both 2L reaction kettles, and the processing kettle C131 is a 15L conventional pilot scale reaction kettle; the number n of the micro-reaction groups 210 is 4; wherein the length of each thermostatic tube (A114, B124 and C134) is 0.18m, and the diameter thereof is 2.5 mm; the diameter of the delay tube 213 is 4mm, and the length is 2 m; the equivalent diameter of the section of the micro-channel reactor 211 is 5mm, and the length of the micro-channel is 1.2 m; the micromixer 212 has a plurality of microchannels having a cross-sectional equivalent diameter of 0.5mm and a microchannel length of 0.7 m.

1) preparation of oil phase fluid a: adding 80g of lecithin and phosphatidic acid, 40g of cholesterol and cholesterol polyoxyethylene ether sodium sulfate (M is 2400), 10g of tripalmitin, 3g of tricaprylin and 0.05g of vitamin C into 1245g of chloroform ethyl ether solution (V: V is 1:1) in advance into a treatment kettle A111, stirring for dissolving, and keeping the temperature at 0-38 ℃ to obtain fluid A for later use;

preparation of inner aqueous phase fluid B: adding a 4 wt% glucose aqueous solution into the treatment kettle B121 in total 1385g, and keeping the temperature at 0-38 ℃ to serve as a fluid B for later use;

preparation of external aqueous phase fluid C: PBS buffer solution with pH of 8.0, L-lysine aqueous solution, glucose aqueous solution and cason aqueous solution are added into a treatment kettle C131 in advance, 5403g of mixed solution with L-lysine concentration of 40mmol/L, glucose concentration of 4 wt% and cason concentration of 0.025 wt% is obtained by mixing finally, and the mixed solution is named as fluid C for standby after heat preservation at 0-38 ℃.

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212, then enters the delay tube 213, and then continues to enter the next 2 micro reaction groups 210 for mixing; during the period, the temperature of the micro reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro reaction unit 200 was adjusted to be maintained between 1 and 30.0MPa by a back pressure valve 300; finally, the residence time of the colostrum W/O fluid D was set to 500s, and the grain size D10, D50, D90 and Span were measured to be 0.43 μm, 4.8 μm, 5.47 μm and 1.05, respectively.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:3.9 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreactor unit 200 was maintained at 1-30MPa by the back pressure valve 300, the residence time of the multiple emulsion W/O/W fluid E was 15s by the adjustment of the metering pumps in conjunction with the length of the delay tube 213, the particle size D10 of the multiple emulsion W/O/W fluid E was 0.42 μm, the particle size D50 was 4.4 μm, the particle size D90 was 4.90 μm, and the Span was 1.02 by sampling.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, the fluid E collected in the collection kettle 400 is introduced into a finished product kettle 610, nitrogen is introduced for solvent removal, under the action of a condenser 620, the solvent is removed, the diluted finished product enters a solvent collection tank 630 for storage, and the polycystic liposome with the solid content of 2% is obtained after detection, wherein the particle size D10 is 0.49 μm, the particle size D50 is 5.1 μm, the particle size D90 is 5.95 μm, and the particle size Span is 1.07, namely 8A; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Example 9

The apparatus for preparing multivesicular liposomes in this example is shown in FIG. 1, and is the same as in example 1;

1) the preparation of the oil phase fluid a, the preparation of the inner aqueous phase fluid B and the preparation of the outer aqueous phase fluid C are the same as in example 1;

2) the fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212 and then enters the delay tube 213, and then continues to enter the next 3 micro reaction groups 210 for mixing; during this period, the temperature of the micro-reaction unit 200 was controlled at 15 ℃ by means of a water bath, the flow rates of the fluid A and the fluid B were adjusted by means of a metering pump to 1:1, and the pressure in the micro-reaction unit 200 was adjusted by means of a back pressure valve 300 to be maintained at about 26MPa (25-27 MPa); finally, the residence time of the colostrum W/O fluid D in the micro-reaction unit 200 was set to 300s (this step is performed as in example 1 except for the collision pressure). Sampling and detecting D particle diameter D10 of the colostrum W/O fluid to be 0.53 μm, D50 to be 5.3 μm, D90 to be 5.9 μm and Span to be 1.01.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled to 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:1.95 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreaction unit 200 was adjusted to about 26MPa (25-27MPa) by the back pressure valve 300, and the residence time of the double emulsion W/O/W fluid E was 15s by the adjustment of the metering pumps in cooperation with the length of the delay tube 213 (this step was conducted, except for the collision pressure, the rest was the same as in example 1). The particle size D10 of the W/O/W fluid E was 0.51 μm, D50 was 5.2 μm, D90 was 5.8 μm, and Span was 1.01 by sampling test.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, introducing the fluid E collected in the collection kettle 400 into a finished product kettle 610, simultaneously introducing nitrogen for solvent removal, under the action of a condenser 620, removing the solvent, and storing the diluted finished product in a solvent collection tank 630, and detecting to obtain the multivesicular liposome with the solid content of 2%, wherein the particle size D10 is 0.54, the particle size D50 is 5.3 μm, the particle size D90 is 6.0 μm, the particle size Span is 1.03, and the name is 9A; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Comparative example 1

The preparation apparatus of multivesicular liposomes provided in this comparative example is substantially the same as that of example 8, except that conventional high-speed shearing equipment is still used to prepare the primary emulsion W/O and the multiple emulsion W/O/W, and specifically, the conventional high-speed shearing equipment is used to replace the micro-reaction unit.

In this comparative example, the preparation method of multivesicular liposomes specifically comprises the following steps:

1) the preparation of the oil phase fluid a, the preparation of the inner aqueous phase fluid B and the preparation of the outer aqueous phase fluid C were the same as in example 8.

2) And introducing the fluid A and the fluid B into a traditional high-speed shearing device for mixing, controlling the temperature at 15 ℃ through a water bath, shearing at the rotating speed of 16000rpm for 9min to form a water-in-oil colostrum fluid D, and sampling to detect that the W/O particle size D10 of the colostrum is 3.2 mu m, the D50 is 9..2 mu m, the D90 is 15.3 mu m and the Span is 1.31.

3) Transferring the fluid D treated by the step (1) into the fluid C, and shearing the fluid C for 20s by using a conventional high-speed shearing device at 4000rpm to form a multiple emulsion of the multivesicular liposome, wherein the multiple emulsion has the particle size D10 of 3.9 microns, the particle size D50 of 9.1 microns, the particle size D90 of 14.8 microns and the particle size Span of 1.19.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, the fluid E collected in the collection kettle 400 is introduced into a finished product kettle 610, nitrogen is introduced for solvent removal, under the action of a condenser 620, the solvent is removed, the diluted finished product enters a solvent collection tank 630 for storage, and the polycystic liposome with the solid content of 2% is obtained after detection, wherein the particle size D10 is 4.1 μm, the particle size D50 is 9.3 μm, the particle size D90 is 15.6 μm, and the particle size Span is 1.23, and the name of the polycystic liposome is comparative example 1B; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Comparative example 2

The apparatus for preparing multivesicular liposomes in this comparative example was the same as in example 8.

In this example, the preparation method of multivesicular liposomes is substantially the same as that of example 8, except that the sterol-based substance selected as the raw material for preparing multivesicular liposomes is only sterol, and no sterol derivative is present. The method comprises the following specific steps:

1) preparation of oil phase fluid a: adding 80g lecithin and phosphatidic acid, 40g cholesterol, 10g tripalmitin, 3g tricaprylin and 5.32g vitamin into a treating kettle A111, dissolving in 1245g chloroform ethyl ether solution (V: V is 1:1), stirring for dissolving, and keeping the temperature at 0-38 deg.C to obtain fluid A;

the preparation of the inner aqueous phase fluid B and the preparation of the outer aqueous phase fluid C were the same as in example 8.

2) The procedure was as in example 8, and finally the residence time of colostrum W/O fluid D was 500s, the D particle size D10 of colostrum W/O fluid was 0.61 μm, D50 was 5.5 μm, D90 was 6.3 μm, and Span was 1.03 as measured by sampling.

3) The procedure was as in example 8, and finally the particle size D10 of the W/O/W fluid E was 0.62 μm, D50 was 5.4 μm, D90 was 6.2 μm, and Span was 1.03.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, the fluid E collected in the collection kettle 400 is introduced into a finished product kettle 610, nitrogen is introduced for solvent removal, under the action of a condenser 620, the solvent is removed, the diluted finished product enters a solvent collection tank 630 for storage, and the polycystic liposome with the solid content of 2% is obtained after detection, wherein the particle size D10 is 0.61 μm, the particle size D50 is 5.6 μm, the particle size D90 is 6.4 μm, and the particle size Span is 1.03, and the name of the polycystic liposome is comparative example 2B; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Comparative example 3

In this example, the preparation of multivesicular liposomes was substantially the same as in example 8, except that the control of the pressure in the steps (especially the pressure in the microreaction cells upon collision of the fluid A, B) was specific:

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212, then enters the delay tube 213, and then continues to enter the next 2 micro reaction groups 210 for mixing; during this period, the temperature of the micro-reaction unit 200 was controlled at 15 ℃ by means of a water bath, the flow rates of fluid A and fluid B were adjusted to 1:1 by means of a metering pump, and the pressure in the micro-reaction unit 200 was adjusted by means of a back pressure valve 300 to be maintained between 35 MPa; finally, the residence time of the colostrum W/O fluid D is 500s, and the grain size D10 of the colostrum W/O fluid D is 0.21 μm, D50 is 1.04 μm, D90 is 3.2 μm and Span is 2.92 by sampling detection.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:3.9 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreactor unit 200 was maintained at 35MPa by the back pressure valve 300, the residence time of the multiple emulsion W/O/W fluid E was 15s by the adjustment of the metering pumps in conjunction with the length of the delay tube 213, the particle diameter D10 of the multiple emulsion W/O/W fluid E was 0.23 μm, D50 was 1.1 μm, D90 was 3.4 μm, and Span was 2.88 by sampling.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, the fluid E collected in the collection kettle 400 is introduced into a finished product kettle 610, nitrogen is introduced for solvent removal, under the action of a condenser 620, the solvent is removed, the diluted finished product enters a solvent collection tank 630 for storage, and the polycystic liposome with the solid content of 2% is obtained after detection, wherein the particle size D10 is 0.49 μm, the particle size D50 is 1.5 μm, the particle size D90 is 4.1 μm, and the particle size Span is 2.4, and the name of the polycystic liposome is comparative example 3B; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Comparative example 4

2) The fluid A and the fluid B respectively pass through respective metering pumps (a metering pump A112 and a metering pump B122) and then respectively enter a microchannel reactor 211 in a first micro-reaction group 210 through respective thermostatic tubes (a thermostatic tube A114 and a thermostatic tube B124), and are mixed to generate colostrum W/O as fluid D; subsequently, the fluid D continues to enter the micro mixer 212, then enters the delay tube 213, and then continues to enter the next 2 micro reaction groups 210 for mixing; during this period, the temperature of the micro-reaction unit 200 was controlled at 15 ℃ by a water bath, the flow rates of the fluid A and the fluid B were adjusted to 1:1 by a metering pump, and the pressure in the micro-reaction unit 200 was adjusted by a back pressure valve 300 to be maintained between 0.5 MPa; finally, the residence time of the colostrum W/O fluid D was set to 500s, and the grain size D10, D50, D90 and Span of the colostrum W/O fluid D were measured to be 1.2 μm, 8.9 μm, 17.8 μm and 1.86 respectively.

3) Continuously introducing the fluid D and the fluid C into the last micro-reaction group 200 for mixing treatment to prepare multiple emulsion W/O/W, wherein the multiple emulsion W/O/W is used as a fluid E; meanwhile, the temperature of the microchannel reactor 212 was controlled at 15 ℃ by a water bath, while the flow rates of the fluid D and the fluid C were adjusted to 1:3.9 by the metering pumps (metering pump A112, metering pump B122, metering pump C132), the pressure in the microreactor unit 200 was maintained at 0.5MPa by the back pressure valve 300, the residence time of the multiple emulsion W/O/W fluid E was 15s by the adjustment of the metering pumps in conjunction with the length of the delay tube 213, the particle size D10 of the multiple emulsion W/O/W fluid E was 1.1 μm, the particle size D50 was 8.6 μm, the particle size D90 was 16.9 μm, and the Span was 1.82.

4) Under the condition of heat preservation, introducing the fluid E into a collection kettle 400 for collection; subsequently, the fluid E collected in the collection kettle 400 is introduced into a finished product kettle 610, nitrogen is introduced for solvent removal, under the action of a condenser 620, the solvent is removed, the diluted finished product enters a solvent collection tank 630 for storage, and the polycystic liposome with the solid content of 2% is obtained after detection, wherein the particle size D10 is 1.4 μm, the particle size D50 is 9.1 μm, the particle size D90 is 18.2 μm, and the particle size Span is 1.81, and the name of the polycystic liposome is comparative example 4B; under the action of the vacuum pump 640, the treated tail gas enters the tail gas collecting device 650.

Example 10

The multivesicular liposomes prepared in the above examples 1A to 9A and comparative examples 1B to 4B have technical effects in the field of application of pesticide preparations.

Particle size and span detection

The particle size and Span of multivesicular liposomes obtained by the preparation method using continuous flow of multivesicular liposomes (examples 1 to 9 and comparative examples 2 to 4) and the conventional high shear apparatus (comparative example 1) after removal of the organic solvent are shown in Table 1.

Span＝(D90-D10)/D50；

Where Span is a Span reflecting the degree of width of the particle size normal distribution diagram, and the smaller Span, the more uniform and narrower the particle size distribution.

TABLE 1 particle size and Span of multivesicular liposomes prepared in examples 1-8 and comparative examples 1-3 after removal of organic solvent

From table 1, the following points can be seen:

firstly, the preparation device of the multivesicular liposome provided by the invention utilizes a continuous flow microchannel reactor to replace the traditional high-speed shearing equipment, and the grain diameter of the multivesicular liposome prepared by adopting continuous flow is more uniform than that of the multivesicular liposome prepared by the traditional high-speed shearing.

Secondly, the Span of the multivesicular liposome prepared in comparative examples 3B and 4B is 2.4 and 1.81 (working pressure is 35MPa and 0.5MPa, respectively), which indicates that if the working pressure is too high or too low (less than 1MPa or more than 30MPa), the particle size distribution of the multivesicular liposome is greatly affected during the preparation process of the multivesicular liposome, and the appropriate process pressure should be selected according to actual conditions.

Second, liquid medicine tank mixing stability experiment

Testing the medicament: 20% of dinotefuran suspending agent, 350g/L of imidacloprid suspending agent, 10% of quizalofop-p-ethyl emulsion in water, 11% of abamectin and etoxazole suspending agent, 50% of thiophanate-methyl wettable powder and 10% of cyhalofop-butyl emulsifiable concentrate.

The test method comprises the following steps: in the examples of the present invention, the adjuvants 1A to 9A, the comparative adjuvants 1A to 4A, and the commercially available adjuvant (hereinafter abbreviated as "commercially available B") were diluted in a tank-mix manner by the two-stage dilution method of agricultural chemicals according to the dilution ratios shown in table 2, respectively; and (3) placing the barrel-mixed liquid medicine into a measuring cylinder with a tip bottom and a plug, standing for 1h, observing whether the barrel-mixed liquid medicine has floating cream precipitation, and recording that the barrel-mixed liquid medicine is qualified if the barrel-mixed liquid medicine has no floating cream precipitation, otherwise, recording that the barrel-mixed liquid medicine is unqualified.

The results are shown in Table 2.

TABLE 2 liquid medicine tank mixing stability experiment

As is evident from the data in table 2:

firstly, after the auxiliary agents 1A to 9A in the embodiment of the invention and the comparative examples 1B to 4B are tank-mixed with various preparations, the difference of the tank-mixing compatibility stability is large, wherein the auxiliary agents 1A to 9A in the embodiment can obtain good compatibility with most pesticide preparation formulations, and the varieties of the formulation with qualified tank-mixing stability of the comparative examples 1B to 4B and the commercial auxiliary agent Anhuole are less than that of the auxiliary agent synthesized by the invention.

Secondly, as can be seen from the comparative example 2B, the sterol derivative is used for replacing the traditional sterol, so that the electronegativity and the steric hindrance of the vesicle are improved, the coagulation of the multivesicular liposome is effectively inhibited, the drug release time of the multivesicular liposome is prolonged, and the drug effect is improved.

Third, field control experiment I

The control object is: cotton aphid Aphis gossypii Glover (piercing-sucking mouthpart pest);

test work: cotton;

medicament: 20% dinotefuran OD (dispersible oil suspension);

the test method comprises the following steps: the adjuvants 1A to 9A of the present invention, the comparative examples 1A to 4A, the commercially available adjuvant ampelox and 20% dinotefuran OD were diluted in a tank mix according to the method described in GBT 17980.75-2004 "Standard on pesticide field efficacy test (II), part 75 of insecticide control Cotton aphid".

The results are shown in Table 3.

TABLE 3 field efficacy test for control of Aphis gossypii Glover (piercing-sucking mouthpart pests)

Serial number	Barrel-mixing combination	Dilution factor	3d control effect (%)
				1	20% dinotefuran OD +1A	400×+1000×	92.45
2	20% dinotefuran OD +2A	400×+1000×	96.87
				3	20% dinotefuran OD +3A	400×+1000×	95.32
4	20% dinotefuran OD +4A	400×+1000×	95.27
				5	20% dinotefuran OD +5A	400×+1000×	93.73
6	20% dinotefuran OD +6A	400×+1000×	95.27
				7	20% dinotefuran OD +7A	400×+1000×	87.56
8	20% dinotefuran OD +8A	400×+1000×	100
				9	20% dinotefuran OD +9A	400×+1000×	98.73
10	20% dinotefuran OD + comparative example 1B	400×+1000×	81.63
				11	20% dinotefuran OD + comparative example 2B	400×+1000×	95.3
12	20% dinotefuran OD + comparative example 3B	400×+1000×	75.21
				13	20% dinotefuran OD + comparative example 4B	400×+1000×	73.35
14	20% dinotefuran OD + commercial B	400×+3000×	79.84
				15	20% dinotefuran OD	400×	72.19
16	Blank Control (CK)

As can be seen from table 3: firstly, the auxiliary agents 1A-9A in the field experiments in the embodiment have synergistic effect on the control of cotton aphids by 20% dinotefuran OD to different degrees, and are superior to the auxiliary agents in comparative examples 1B, 3A and 4A and the commercial auxiliary agent Anmelt.

Second, the control effect of example 8A is better than that of comparative example 2B, which shows that the effect of adding the catalpol derivative is better than that of the sterol substance, and the drug effect can be effectively increased.

Third, the control effect of example 8A is superior to that of comparative examples 3B and 4B, which illustrates that the uniformity of the particle size of the multivesicular liposome prepared within the process pressure range is superior to that of the multivesicular liposome prepared outside the process pressure; the prevention effect of A9 is better than that of 1A-7A, and the grain diameter uniformity of the multivesicular liposome prepared in the process pressure range is better than that of the multivesicular liposome prepared outside the process pressure;

when the multivesicular liposome is applied to the field of pesticides as an active substance for wrapping pesticides, the quality of pesticide effect has a great relationship with particle size distribution and particle size, if the particle size of the multivesicular liposome is too large/too small, the particle size distribution is in wide distribution, so that the encapsulation of a raw pesticide and the conduction and absorption of the pesticide are not facilitated, a stable preparation can be difficult to form, the capability of transporting and carrying active ingredients of the pesticide can be relatively weak, and the carried raw pesticide can hardly penetrate through a target to play a role in increasing the conduction and absorption of the raw pesticide.

Third, field control experiment two

The control object is: rice field Euphorbia lathyris Leptochloa chinensis (L.) Nees (Gramineae Euphorbia weed);

test work: rice; medicament: 10% cyhalofop-butyl EC (emulsifiable concentrate);

the test method comprises the following steps: according to the method of GB/T17980.40-2000 method for controlling postemergence herbicide stem and leaf treatment of paddy fields in part 40 of the criteria of pesticide field efficacy test, the adjuvants 1A-9A of the examples and the adjuvants 1A-4A of the comparative examples of the invention, the commercially available adjuvant clotrimazole and 10% cyhalofop-butyl EC were used in a tank-mix dilution manner, and the results are shown in Table 4.

TABLE 4 field efficacy test for control of rice field Euphorbia lathyris Leptochloa chinensis (L.) Nees

Serial number	Barrel-mixing combination	Dilution factor	7d control effect (%)
				1	10% Cyhalofop-butyl EC +1A	150×+1000×	89.75
2	10% Cyhalofop-butyl EC +2A	150×+1000×	96.27
				3	10% Cyhalofop-butyl EC +3A	150×+1000×	93.36
4	10% cyhalofop-butyl EC +4A	150×+1000×	95.86
				5	10% Cyhalofop-butyl EC +5A	150×+1000×	94.96
6	10% Cyhalofop-butyl EC +6A	150×+1000×	90.38
				7	10% cyhalofop-butyl EC +7A	150×+1000×	68.28
8	10% Cyhalofop-butyl EC +8A	150×+1000×	98.78
				9	10% cyhalofop-butyl EC +9A	150×+1000×	98.62
10	10% Cyhalofop-butyl EC + control 1A	150×+1000×	69.07
				11	10% Cyhalofop-butyl EC + control 2A	150×+1000×	94.12
12	10% Cyhalofop-butyl EC + control 3A	150×+1000×	75.12
				13	10% Cyhalofop-butyl EC + control 4A	150×+1000×	60.12
14	10% Cyhalofop-butyl EC + commercially available B	150×+1000×	74.43
				15	10% cyhalofop-butyl EC	150×	72.29
16	CK

As can be seen in Table 4, in the field test, the adjuvants 1A to 9A in the examples have different degrees of synergistic effect on 10% cyhalofop-butyl EC control Euphorbia lathyris, and are superior to the adjuvants 1B, 3B and 4B and the commercially available adjuvant Anmelt.

Second, example 8A is superior to comparative example 2B in control effect, which indicates that the addition of catalpol derivatives is superior to sterols.

Fifth, field control experiment III

The control object is: berengeriana f.sp.piricola, a berlingeriana f.sp.piricola; test work: apple, apple;

medicament: 50% thiophanate methyl WP (wettable powder);

the test method comprises the following steps: according to GBT 17980.118-2004 part 118 of pesticide field efficacy test criteria (II): the results of using the adjuvants 1A to 9A of the present invention and the adjuvants 1A to 4A of the comparative examples, and the commercially available adjuvant Anmelt with 50% thiophanate-methyl WP in a tank-mix dilution method described in the bactericide prevention and treatment of apple ring spot are shown in Table 5.

TABLE 5 field efficacy test for controlling apple ring spot

Serial number	Barrel-mixing combination	Dilution factor	Control effect (%)
				1	50% thiophanate-methyl WP +1A	500×+1000×	97.21
2	50% thiophanate methyl WP +2A	500×+1000×	97.56
				3	50% thiophanate-methyl WP +3A	500×+1000×	98.78
4	50% thiophanate-methyl WP +4A	500×+1000×	94.43
				5	50% thiophanate-methyl WP +5A	500×+1000×	98.04
6	50% thiophanate-methyl WP +6A	500×+1000×	94.37
				7	50% thiophanate-methyl WP +7A	500×+1000×	88.41
8	50% thiophanate-methyl WP +8A	500×+1000×	99.89
				9	50% thiophanate-methyl WP +9A	500×+1000×	99.62
10	50% thiophanate-methyl WP + control 1A	500×+1000×	49.54
				11	50% thiophanate-methyl WP + control 2A	500×+1000×	95.32
12	50% thiophanate-methyl WP + control 3A	500×+1000×	48.63
				13	50% thiophanate-methyl WP + control 4A	500×+1000×	70.68
14	50% thiophanate methyl WP + commercially available B	500×+1000×	56.68
				15	50% thiophanate methyl WP	500×	76.23
16	CK

As can be seen from table 5: firstly, in the field test, the auxiliary agents 1A-9A in the embodiment have synergistic effect on the prevention and treatment of apple ring spot by 50% thiophanate methyl WP, and are superior to comparative examples 1B, 3B and 4B and commercial auxiliary agent Anhuile.

when the multivesicular liposome is applied as an active substance for wrapping pesticides, the quality of the pesticide effect has a great relationship with the size distribution of the particle size of the vesicles in the multivesicular liposome, if the particle size is too large/small and the particle size distribution is wide, the wrapping of the original drugs and the conduction and absorption of the drugs are not facilitated, a stable preparation can be difficult to form, the capacity of transporting and carrying active ingredients of the pesticide can be relatively weak, and the carried original drugs can hardly penetrate through a target to play a role in increasing the conduction and absorption of the original drugs.

Claims

1. A blank multivesicular liposome comprises preparation auxiliary materials, a solvent and raw materials, and is characterized in that the preparation raw materials comprise the following components in parts by weight:

40-80 parts of phospholipid substances;

10-40 parts of sterol substances;

5-10 parts of neutral liposome;

1-5 parts of fatty acid ester substances;

0.01-0.05 parts of vitamin.

2. The multivesicular liposome of claim 1, wherein said phospholipid material is a phospholipid and/or a hydrogenated derivative of a phospholipid; the sterol substance is sterol and/or sterol derivatives.

3. A preparation device of blank multi-vesicular liposome comprises a feeding unit, a micro-reaction unit, a collection kettle and a solvent removal unit which are sequentially communicated, and is characterized in that a communication pipeline between the micro-reaction unit and the collection kettle is also provided with a back pressure valve; wherein the content of the first and second substances,

the micro-reaction unit comprises a plurality of micro-reaction groups which are connected in series, and each micro-reaction group comprises a micro-channel reactor, a micro-mixer and a time delay tube which are sequentially communicated; the delay tube of the former micro-reaction group is communicated with the micro-channel reactor of the latter micro-reaction group;

the feeding unit comprises a treatment kettle and a metering pump which are sequentially communicated, and the treatment kettle of the feeding unit is communicated with the micro-channel reactor of the micro-reaction unit through the metering pump.

4. The apparatus for preparing multivesicular liposomes according to claim 3, wherein the microchannel reactor has microchannels having a cross-sectional equivalent diameter of 0.01 to 10mm and a length of 0.2 to 2 m;

the micro mixer is provided with a micro channel, the equivalent diameter of the cross section of the micro channel is 0.01-10 mm, and the length of the micro channel is 0.4-8.5 m;

the diameter of the channel of the delay tube is 1-5 mm, and the length of the channel is 0.5-15 m.

5. A method for preparing blank multivesicular liposomes, comprising the steps of:

1) preparing an oil phase as a fluid A, preparing an inner water phase as a fluid B and preparing an outer water phase as a fluid C;

2) mixing fluid A, B by alternately using a microchannel reactor and a micromixer until primary emulsion W/O with the particle size D50 of 1-6 mu m and Span of 1.01-1.07 is prepared, wherein the primary emulsion W/O is used as fluid D;

3) mixing fluid C, D by using a continuous flow microchannel reactor and a micro mixer in sequence to prepare compound emulsion W/O/W serving as fluid E;

4) and (3) carrying out solvent removal treatment on the fluid E to obtain a blank multivesicular liposome.

6. The method for preparing multivesicular liposomes according to claim 5, wherein in step 1), the fluid A is prepared by dissolving phospholipid substances, sterol substances, neutral liposomes, vitamins and fatty acid ester substances in a solvent, wherein the mass fraction of the solvent is 75-90%;

7. The method for preparing multivesicular liposomes according to claim 5 or 6, wherein in the step 2), the collision pressure P is 1 to 30MPa and the collision flow rate is 1 (0.1 to 1) when the fluid A and the fluid B are mixed.

8. The method for preparing multivesicular liposomes according to claim 7, wherein the total residence time t1 of the fluid D in the micro reaction unit in the step 2) is 60 to 900 s.

9. The method for preparing multivesicular liposomes according to claim 7, wherein the collision pressure P in the mixing of the fluid C and the fluid D in the step 3) is 1 to 30MPa and the collision flow rate is 1 (1 to 10).

10. The method for preparing multivesicular liposomes according to claim 9, wherein the total residence time t2 of the fluid E in the micro reaction group in the step 3) is 10 to 15 s.