CN114887562B - Preparation method of liposome - Google Patents

Abstract

The invention provides a preparation method of liposome, and aims to solve the technical problem of low preparation rate of the existing liposome preparation method. The adopted technical scheme is as follows: a method for preparing a liposome comprising the steps of: s1: dissolving lipid material and lipid-soluble material to be encapsulated in organic solvent to obtain oil phase; dissolving stabilizer in water to obtain external water phase; s2: flowing the outer aqueous phase along a first channel; enabling the oil phase to flow along the first micro-channel and vertically enter the first channel from the outlet end of the first micro-channel; the oil phase is allowed to diffuse into the outer aqueous phase to form liposomes. The oil phase and the external water phase are respectively switched between a flowing state and a flowing stopping state in a rapid staggered manner. The invention can break the flow rate limit of the oil phase and improve the preparation rate of the liposome.

Description

Preparation method of liposomes

Technical field

The present invention relates to the technical field of liposome production, and specifically to a preparation method of liposomes.

Background technique

When amphiphilic molecules such as phospholipids and sphingolipids are dispersed in the internal water phase, the hydrophobic tails of the molecules tend to cluster together and avoid the internal water phase, while the hydrophilic heads are exposed to the internal water phase, forming a bilayer structure. Closed vesicles called liposomes. As a carrier, liposomes are widely used in cosmetics, skin care products, clinical medicine and other fields.

The application of microfluidic technology to prepare liposomes is a current research hotspot. A common method for preparing liposomes using microfluidic technology is to allow the oil phase that dissolves the substance to be encapsulated to flow along the microchannel, and to allow the external aqueous phase to flow through the outlet end of the microchannel, thereby processing the oil phase flowing out of the microchannel. Squeezing and enveloping, the oil phase is dispersed into the external water phase to form liposomes.

Most of the existing technologies use micro-injection pumps to provide power for the oil phase and the external water phase, so that the oil phase and the external water phase meet at a substantially constant speed. When using this method to prepare liposomes; in order for the oil phase to be better dispersed into the external aqueous phase to form liposomes instead of forming a stratified fluid parallel to the external aqueous phase, the flow rate of the oil phase needs to be limited. It is extremely slow and has the disadvantages of low preparation throughput and slow preparation rate.

Contents of the invention

The object of the present invention is to provide a method for preparing liposomes. It causes the oil phase to quickly switch between the flowing state and the stopped flowing state, and intermittently diffuses into the external aqueous phase to form liposomes. This can effectively avoid the formation of stratified fluids parallel to the external water phase when the oil phase flows rapidly, breaks the flow rate limit of the oil phase, and improves the liposome preparation throughput and preparation rate.

specifically,

The preparation method of liposomes includes the following steps:

S1: Dissolve the lipid substance and the fat-soluble substance to be encapsulated in an organic solvent to form an oil phase; dissolve the stabilizer in water to form an external aqueous phase;

S2: Make the external water phase flow along the first channel; make the oil phase flow along the first microchannel, and vertically enter the first channel from the outlet end of the first microchannel; make the oil phase diffuse into the external water phase to form liposomes;

Among them, the oil phase and the external water phase quickly switch between the flowing state and the stopped flow state respectively; the oil phase is in a flowing state, and when it overflows from the outlet end of the first microchannel, the external water phase is in a stopped flowing state; the oil phase is in a stopped state. In the flowing state, the external water phase is in a flowing state and flows along the first right channel with the overflowing oil phase.

The working principle of the present invention is as follows: make the oil phase flow along the first microchannel and vertically enter the first channel from the outlet end of the first microchannel; make the external water phase flow along the first channel. The oil phase and external water phase are alternately switched between the flowing state and the stopped flowing state quickly. When the oil phase is in a flowing state, the external water phase is in a stopped flowing state, so that the oil phase can more easily overflow from the outlet end of the first microchannel. When the oil phase stops flowing, the external aqueous phase is in a flowing state and will carry the overflowing oil phase and flow along the first right channel, thereby causing the oil phase to disperse in the external aqueous phase to form liposomes.

It can be seen from this that the beneficial effects of the present invention are: causing the oil phase to overflow intermittently and diffusing into the external water phase to form liposomes; it can effectively avoid the formation of stratified fluids juxtaposed with the external water phase when the oil phase flows rapidly, breaking the The flow rate limitation of the oil phase is conducive to improving the preparation throughput and preparation rate of liposomes.

Optionally, the lipid substance is one or more of phospholipids, cholesterol, and amphoteric substances; the organic solvent is ether, ethanol, cyclohexane or chloroform; the stabilizer is a monosaccharide or an oligosaccharide. , one or more of acids and bases.

Optionally, the first microchannel is provided in a first microtube, and the first microtube has a plurality of first microchannels in parallel; the first microtube is limited to a first microtubule chamber, and the first microtube is A microtube chamber has a plurality of first limiting through holes for accommodating first microtubes; the first microtube chamber is connected to an oil phase pump that intermittently transports oil phase.

Optionally, the first channel is provided in the first manifold; the first channel runs through the first manifold in the front-to-back direction, and both ends are respectively connected with the external water phase pump that intermittently transports the external water phase; the third channel A microtube chamber is located on the left side of the first channel, and the two are connected through the first microtube limited to the first limiting through hole; the first manifold has a first right channel penetrating from the first channel to the right; The first microtubes are distributed on the front and rear sides of the first right channel; the left and right width of the first channel is less than or equal to 1 mm.

The invention also provides another method for preparing liposomes, which includes the following steps:

S1: Dissolve lipid substances in organic solvents, or dissolve lipid substances and fat-soluble substances to be encapsulated in organic solvents to make an oil phase; dissolve water-soluble substances to be encapsulated in water to make internal water phase; dissolve the stabilizer in water to make the external water phase;

S2: Make the oil phase flow along the first channel; make the internal water phase flow along the first microchannel, and enter the first channel vertically from the outlet end of the first microchannel; prepare a mixed phase with diffused water bubbles;

S3: Make the external water phase flow along the second channel; make the mixed phase enter the second microchannel along the first right channel, and vertically enter the second channel from the outlet end of the second microchannel; make the mixed phase diffuse into the external water phase to form Liposomes;

Among them, the internal water phase, oil phase, and external water phase quickly switch between the flowing state and the stopped flowing state respectively; when the internal water phase is in the flowing state, the oil phase and the external water phase are in the stopped flowing state; when the oil phase is in the flowing state, , the inner water phase and the outer water phase are in a stopped flowing state; when the outer water phase is in a flowing state, the inner water phase and the oil phase are in a stopped flowing state.

Optionally, a stabilizer is dissolved in the internal water phase; the stabilizer is one or more of monosaccharides, oligosaccharides, acids, and alkali; the lipid substance is phospholipid, cholesterol, and amphoteric substances. One or more of; the organic solvent is ether, ethanol, cyclohexane or chloroform.

Optionally, the first microchannel is provided in a first microtube, and the first microtube has a plurality of first microchannels in parallel; the first microtube is limited to a first microtubule chamber, and the first microtube is A microtube chamber has a plurality of first limiting through holes for accommodating first microtubes; the first microtube chamber is connected to an internal water phase pump that intermittently transports internal water phase.

Optionally, the first channel is provided in the first manifold; the first channel runs through the first manifold in the front-to-back direction, and both ends are respectively connected with oil phase pumps that intermittently transport the oil phase; the first micro The tube chamber is located on the left side of the first channel, and the two are connected through the first microtube limited to the first limiting through hole; the first manifold has a first right channel penetrating from the first channel to the right; the The first microtubes are distributed on the front and rear sides of the first right channel; the left and right width of the first channel is less than or equal to 1 mm.

Optionally, the second microchannel is provided in a second microtube, and the second microtube has a plurality of second microchannels in parallel; the second microtube is limited to a second microtubule chamber, and the second microtube has a plurality of parallel second microchannels. The second microtube chamber has a plurality of second limiting through holes for accommodating the second microtube; the second microtubule chamber is connected with the first right channel.

Optionally, the second channel is provided in the second manifold; the second channel runs through the second manifold in the up and down direction, and both ends are respectively connected with the external water phase pump that intermittently transports the external water phase; the second channel The two microtube chambers are located on the left side of the second channel, and the two are connected through the second microtube limited to the second limiting through hole; the second manifold has a second right channel penetrating from the second channel to the right; The second microtubes are distributed on the upper and lower sides of the second right channel; the left and right width of the second channel is less than or equal to 1 mm.

The working principle of the present invention is: the internal water phase pump drives the internal water phase to flow along the first microchannel, and at the same time, the oil phase and the external water phase stop flowing; then the external water phase pump drives the external water phase to flow along the second channel, and at the same time Stop the flow of the inner water phase and the oil phase; then let the oil phase pump drive the oil phase to flow along the first channel, and at the same time stop the flow of the inner water phase and the outer water phase; finally let the outer water phase pump drive the outer water phase to flow along the second channel, At the same time, stop the flow of the internal water phase and oil phase; in this way, repeat the above steps to obtain liposomes. Specifically, when the internal water phase pump drives the internal water phase to overflow from the outlet end of the first microchannel, the mixed phase will also overflow from the outlet end of the second microchannel; then the external water phase pump drives the external water phase to flow along the first microchannel. The two-channel flow allows the external water to squeeze and envelop the overflowing mixed phase, causing the mixed phase to disperse into the external water phase to form liposomes. When the oil phase pump drives the oil phase to flow along the first channel, the oil phase will squeeze and encircle the overflowing internal water phase, causing the internal water phase to disperse into the oil phase to form bubbles; at the same time, the mixed phase will also flow from the second channel. The outlet end of the microchannel overflows; then the external water phase pump drives the external water phase to flow along the second channel, so that the external water can be squeezed and wrapped around the overflowing mixed phase, so that the mixed phase is dispersed into the external water phase to form lipids. plastid.

It can be seen from this that the beneficial effects of the present invention are: causing the internal water phase to overflow intermittently and dispersed into the oil phase to form a mixed phase with diffused blisters; causing the mixed phase to overflow intermittently and disperse into the external water phase to form lipids body. It not only avoids the formation of stratified fluids parallel to the oil phase when the internal water phase flows rapidly, but also avoids the formation of stratified fluids parallel to the external water phase when the mixed phase flows rapidly; breaking the flow rate limit of the internal water phase and oil phase, It is beneficial to improve the preparation throughput and preparation rate of liposomes.

Description of the drawings

In order to explain the embodiments of the present application or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only These are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a graph of the preparation rate-encapsulation rate coordinate curve of Example 1 and Comparative Example 1;

Figure 2 is a graph of the preparation rate-encapsulation rate coordinate curve of Example 2 and Comparative Example 2;

Figure 3 is a schematic diagram of the cooperation of the oil phase pump, the external water phase pump, and the first manifold;

Figure 4 is a schematic structural diagram of the first busbar;

Figure 5 is a schematic diagram of the assembly of the first busbar and the first microtube;

Figure 6 is a schematic structural diagram of the first microtubule;

Figure 7 is a schematic diagram of the cooperation of the internal water phase pump, the oil phase pump, the external water phase pump, the first manifold, and the second manifold;

Figure 8 is a schematic diagram of the connection between the first bus part and the second bus part;

Figure 9 is a schematic assembly diagram of the first busbar and the second busbar;

Figure 10 is a schematic diagram of Figure 9 from another angle;

Figure 11 is a schematic structural diagram of the oil phase pump;

Figure 12 is an assembly diagram of the transmission mechanism;

Figure 13 is a schematic diagram of the connection between the transmission mechanism and the diaphragm;

Figure 14 is a schematic diagram of the assembly of the pump chamber, liquid inlet cavity, and liquid outlet cavity;

Figure 15 is a schematic diagram of the assembly of the liquid inlet chamber;

Figure 16 is a schematic diagram of Figure 15 from another angle;

Reference signs: 1. First microtube; 2. First microtube chamber; 3. First limiting through hole; 4. Oil phase pump; 5. First channel; 6. First manifold; 7. External Water phase pump; 8. First right channel; 9. Internal water phase pump; 10. Second microtube; 11. Second microtubule chamber; 12. Second limiting through hole; 13. Second channel; 14. Second manifold; 15. Second right channel; 16. Pump chamber; 17. Liquid inlet cavity; 18. Liquid outlet cavity; 19. Liquid inlet control; 20. Liquid outlet control; 21. Discharge pipe; 22 , diaphragm; 23. power source; 24. pump cover; 25. limiter; 26. plug rod; 27. eccentric shaft; 28. connecting piece; 29. main shaft part; 30. eccentric part; 31. first closed section ; 32. First circulation section; 33. First main channel; 34. First auxiliary channel; 35. Second closed section; 36. Second circulation section.

Detailed ways

In the following, only certain exemplary embodiments are briefly described. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

In the description of the present invention, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", " "Back", "Left", "Right", "Vertical", "Horizontal", "Top", "Bottom", "Inside", "Outside", "Clockwise", "Counterclockwise", "Axis" The orientation or positional relationship indicated by "radial direction", "circumferential direction", etc. is based on the orientation or positional relationship shown in FIG. 3 or FIG. 7, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying. The devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation and therefore are not to be construed as limitations of the invention.

In addition, the terms “first” and “second” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include one or more of these features. In the description of the present invention, "plurality" means two or more than two, unless otherwise explicitly and specifically limited.

In the present invention, unless otherwise clearly stated and limited, the terms "installation", "connection", "connection", "fixing" and other terms should be understood in a broad sense. For example, it can be a fixed connection or a detachable connection. , or integrated; it can be a mechanical connection, an electrical connection, or a communication; it can be a direct connection, or an indirect connection through an intermediate medium, or an internal connection between two elements or an interaction between two elements . For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

Embodiment 1

S1: Dissolve vitamin A, phospholipid, and cholesterol in chloroform to prepare an oil phase with a vitamin A concentration of 10g/L, a phospholipid concentration of 30g/L, and a cholesterol concentration of 20g/L; dissolve phosphoric acid and glucose in water to prepare The external aqueous phase has a pH of 7.4 and a sugar concentration of 1000mmol/L.

S2: The shilling oil phase pump 4 drives the oil phase to flow along the first microchannel, and at the same time, the external water phase pump 7 stops driving the external water phase to flow.

S3: Then, the external water phase pump 7 is allowed to drive the external water phase to flow along the first channel 5, and the oil phase pump 4 is stopped to drive the oil phase flow at the same time.

S4: Loop steps S2 and S3 at least once per second, and the number of cycles per second is proportional to the traffic per second. Collect the vitamin A liposome solution discharged from the first right channel 8.

Embodiment 2

S1: Dissolve phospholipids and cholesterol in absolute ethanol to make an oil phase with a phospholipid concentration of 50g/L and a cholesterol concentration of 20g/L; dissolve hydrochloric acid and doxorubicin in water to make doxorubicin with a pH of 4. The internal water phase with a concentration of 10g/L; dissolve hydrochloric acid and glucose in water to make an external water phase with a pH of 4 and a sugar concentration of 1200mmol/L.

S2: Shilling the internal water phase pump 9 to drive the internal water phase to flow along the first microchannel; at the same time, the oil phase pump 4 stops driving the oil phase flow, and the external water phase pump 7 stops driving the external water phase flow.

S3: Then, the external water phase pump 7 drives the external water phase to flow along the second channel 13; at the same time, the internal water phase pump 9 stops driving the internal water phase flow, and the oil phase pump 4 stops driving the oil phase flow.

S4: Then let the oil phase pump 4 drive the oil phase to flow along the first channel 5; at the same time, make the inner water phase pump 9 stop driving the inner water phase flow, and make the outer water phase pump 7 stop driving the outer water phase flow.

S5: Finally, the external water phase pump 7 drives the external water phase to flow along the second channel 13; at the same time, the internal water phase pump 9 stops driving the internal water phase flow, and the oil phase pump 4 stops driving the oil phase flow.

S6: Loop steps S2 to S5 at least once per second, and the number of cycles per second is proportional to the traffic per second. Collect the doxorubicin liposome solution discharged from the second right channel 15.

Embodiment 1

S1: Dissolve vitamin A, phospholipid, and cholesterol in chloroform to prepare an oil phase with a vitamin A concentration of 10g/L, a phospholipid concentration of 30g/L, and a cholesterol concentration of 20g/L; dissolve phosphoric acid and glucose in water to prepare The external aqueous phase has a pH of 7.4 and a sugar concentration of 1000mmol/L.

S2: Inject the oil phase into the first microchannel at a constant speed through the oil phase microinjection pump, and inject the external water phase into the first channel 5 through the external water phase microinjection pump at a constant speed. Collect the vitamin A liposome solution discharged from the first right channel 8.

Embodiment 2

S1: Dissolve phospholipids and cholesterol in absolute ethanol to make an oil phase with a phospholipid concentration of 50g/L and a cholesterol concentration of 20g/L; dissolve hydrochloric acid and doxorubicin in water to make doxorubicin with a pH of 4. The internal water phase with a concentration of 10g/L; dissolve hydrochloric acid and glucose in water to make an external water phase with a pH of 4 and a sugar concentration of 1200mmol/L.

S2: Inject the inner water phase into the first microchannel at a constant speed through the internal water phase microinjection pump, inject the oil phase into the first channel 5 at a constant speed through the oil phase microinjection pump, and inject the outer water phase into the second channel 13 at a constant speed through the external water phase microinjection pump. External water phase. Collect the doxorubicin liposome solution discharged from the second right channel 15.

For Example 1 and Comparative Example 1, in addition to the oil phase pump 4 that intermittently delivers the oil phase and the oil phase micro-injection pump that delivers the oil phase at a constant speed, as well as the external water phase pump 7 that intermittently delivers the external water phase and the external water phase that delivers the external water phase at a constant speed. External aqueous phase microinjection pump, other devices adopt the same specifications. The frequency of the oil phase pump 4 and the external water phase pump 7 switching between the delivery state and the stop delivery state per unit time is proportional to the delivery volume per unit time. By adjusting the delivery volume of the oil phase pump 4 and the external water phase pump 7 per unit time, the rate of preparing the vitamin A liposome solution in Example 1 is adjusted; and samples are taken to measure the encapsulation rates corresponding to the different preparation rates of Example 1. . By adjusting the delivery volume per unit time of the oil phase microinjection pump and the external aqueous phase microinjection pump, the rate of preparing the vitamin A liposome solution in Comparative Example 1 was adjusted; and samples were taken to measure the package contents corresponding to different preparation rates in Comparative Example 1. Closure rate. Taking the preparation rate as the abscissa and the encapsulation rate as the ordinate, a coordinate curve chart is made, and the results are shown in Figure 1.

It can be seen from Figure 1 that as the preparation rate increases, the encapsulation rate of the vitamin A liposome solution prepared in Example 1 does not decrease significantly; that is to say, as long as the oil phase pump 4 and the external water phase are increased The frequency at which the pump 7 switches between the delivery state and the stop delivery state within a unit time is the number of times steps S2 and S3 are cycled per unit time; the preparation rate of the vitamin A liposome solution can be increased without any hindrance. On the other hand, in Comparative Example 1, as the preparation rate continues to increase, the encapsulation rate is difficult to maintain at a good level and will continue to decrease.

For Example 2 and Comparative Example 2, in addition to the internal water phase pump 9 that intermittently transports the internal water phase and the internal water phase microinjection pump that transports the internal water phase at a constant speed, the oil phase pump 4 that transports the oil phase intermittently and the internal water phase pump 4 that transports the oil phase at a constant speed The oil phase microinjection pump, as well as the external water phase pump 7 for intermittent delivery of the external water phase and the external water phase microinjection pump for uniform delivery of the external water phase; other devices adopt the same specifications. The frequency at which the internal water phase pump 9, the oil phase pump 4, and the external water phase pump 7 switch between the delivery state and the stop delivery state per unit time is proportional to the delivery volume per unit time. By adjusting the delivery volume of the internal water phase pump 9, the oil phase pump 4, and the external water phase pump 7 per unit time, the rate of preparing the doxorubicin liposome solution in Example 1 is adjusted; and samples are taken to measure the different preparations in Example 1 The encapsulation rate corresponding to the rate. By adjusting the delivery volume per unit time of the internal aqueous phase microinjection pump, the oil phase microinjection pump, and the external aqueous phase microinjection pump, the rate of preparing the doxorubicin liposome solution in Comparative Example 1 was adjusted; and samples were taken to measure the Comparative Example An encapsulation rate corresponding to different preparation rates. Taking the preparation rate as the abscissa and the encapsulation rate as the ordinate, a coordinate curve chart is made, and the results are shown in Figure 2.

It can be seen from Figure 2 that as the preparation rate increases, the encapsulation rate of the doxorubicin liposome solution prepared in Example 1 does not decrease significantly; that is to say, as long as the internal water phase pump 9, oil The frequency at which the phase pump 4 and the external aqueous phase pump 7 switch between the delivery state and the stop delivery state per unit time is the number of times steps S2 to S5 are cycled per unit time; this can increase the efficiency of the doxorubicin liposome solution without any hindrance. preparation rate. On the other hand, in Comparative Example 1, as the preparation rate continues to increase, the encapsulation rate is difficult to maintain at a good level and will continue to decrease.

In summary, the present invention transports the oil phase and the external water phase in a high-frequency alternating manner, or transports the internal water phase, oil phase, and external water phase in a high-frequency alternating manner; breaking the flow rate limit of the internal water phase and the oil phase, It is beneficial to improve the preparation throughput and preparation rate of liposomes.

As shown in Figures 3 to 6, in Embodiment 1 and Comparative Example 1, the first microchannel is provided in the first microtube 1, and the first microtube 1 has multiple first microchannels in parallel; The first microtube 1 is limited to a first microtube chamber 2, and the first microtube chamber 2 has a plurality of first limiting through holes 3 for accommodating the first microtube 1; the first microtube Chamber 2 is connected to an oil phase pump 4 that delivers the oil phase intermittently. It should be understood that the first microtubes 1 can be arranged in multiple columns along the length direction of the first channel 5, and adjacent columns are staggered. In addition, an expanded cap portion can be provided at one end of the first microtube 1 located in the first microtube chamber 2, and a first resistor can be provided in the first microtube chamber 2 to form a tight contact with the cap portion of the first microtube 1. Tighten the tube plate; and a socket can be provided at the outlet of the oil phase pump 4 to form a resistance to the first pressing tube plate.

Further, the first channel 5 is provided in the first manifold 6; the first channel 5 penetrates the first manifold 6 in the front-to-back direction, and both ends are respectively connected with the external water phase pump 7 that intermittently transports the external water phase. ; The first microtube chamber 2 is located on the left side of the first channel 5, and the two are connected through the first microtube 1 limited to the first limiting through hole 3; the first busbar 6 has a 5 runs through the first right channel 8 to the right; the first microtubes are distributed on the front and rear sides of the first right channel 8; the left and right width of the first channel 5 is less than or equal to 1 mm. It should be understood that the preparation throughput can be doubled by allowing the oil phase and the external water phase to flow from the front and rear ends toward the first right channel 8 in the middle instead of flowing unidirectionally from front to back or from back to front.

It should be noted that Example 1 and Comparative Example 1 use the same three-dimensional fluid focusing structure as mentioned above to prepare liposomes; the purpose is to compare high-frequency intermittent delivery of oil phase and external water phase with continuous delivery while controlling a single variable. Advantages and disadvantages of oil phase and external water phase. In fact, when the existing technology uses microfluidic technology to prepare liposomes, most of the first microchannels and the first channel 5 are processed on the same plane; the first microchannel and the first channel 5 are made to be Y-shaped to achieve Confluence of oil phase and external aqueous phase; preparation of liposomes by two-dimensional fluid focusing. In comparison, the three-dimensional fluid focusing used in Example 1 and Comparative Example 1 will greatly increase the production throughput and production rate. In other words, even Comparative Example 1 has the advantage of greatly improving the preparation throughput and preparation rate compared with the existing technology.

As shown in Figures 6 to 10, in Embodiment 2 and Comparative Example 2, the first microchannel is provided in the first microtube 1, and the first microtube 1 has multiple first microchannels in parallel; The first microtube 1 is limited to a first microtube chamber 2, and the first microtube chamber 2 has a plurality of first limiting through holes 3 for accommodating the first microtube 1; the first microtube The chamber 2 is connected to the internal water phase pump 9 which intermittently transports the internal water phase. It should be understood that the first microtubes 1 can be arranged in multiple columns along the length direction of the first channel 5, and adjacent columns are staggered. In addition, an expanded cap portion can be provided at one end of the first microtube 1 located in the first microtube chamber 2, and a first resistor can be provided in the first microtube chamber 2 to form a tight contact with the cap portion of the first microtube 1. Tighten the tube plate; and a socket can be provided at the outlet of the internal water phase pump 9 to form a resistance to the first pressing tube plate.

Further, the first channel 5 is provided in the first manifold 6; the first channel 5 penetrates the first manifold 6 in the front and rear direction, and both ends are respectively connected with the oil phase pump 4 that intermittently transports the oil phase; so The first microtube chamber 2 is located on the left side of the first channel 5, and the two are connected through the first microtube 1 limited to the first limiting through hole 3; the first bus 6 has a direction from the first channel 5 to The first right channel 8 runs right through; the first microtubes are distributed on the front and rear sides of the first right channel 8; the left and right width of the first channel 5 is less than or equal to 1 mm. It should be understood that the preparation throughput can be doubled by allowing the internal water phase and oil phase to flow from the front and rear ends toward the first right channel 8 in the middle instead of flowing unidirectionally from front to back or from back to front.

Further, the second microchannel is provided in the second microtube 10, and the second microtube 10 has a plurality of second microchannels in parallel; the second microtube 10 is limited to the second microtube chamber 11, The second microtube chamber 11 has a plurality of second limiting through holes 12 for accommodating the second microtubes 10; the second microtube chamber 11 is connected to the first right channel 8. It should be understood that the second microtubes 10 can be arranged in multiple columns along the length direction of the second channel 13, and adjacent columns are staggered. In addition, an expanded cap portion can be provided at one end of the second microtube 10 located in the second microtube chamber 11, and a second resistor can be provided in the second microtube chamber 11 to form a tight contact with the cap portion of the second microtube 10. Tighten the tube plate; and a socket can be provided at the outlet of the first right channel 8 to form a resistance to the second tightening tube plate.

Further, the second channel 13 is provided in the second manifold 14; the second channel 13 penetrates the second manifold 14 in the up and down direction, and both ends are respectively connected with the external water phase pump 7 that intermittently transports the external water phase. ; The second microtube chamber 11 is located on the left side of the second channel 13, and the two are connected through the second microtube 10 limited to the second limiting through hole 12; the second manifold 14 has a channel from the second channel 13 runs through the second right channel 15 to the right; the second microtubes 10 are distributed on the upper and lower sides of the second right channel 15; the left and right width of the second channel 13 is less than or equal to 1 mm. It should be understood that the preparation throughput can be doubled by allowing the mixed phase and the external aqueous phase to flow from the upper and lower ends toward the second right channel 15 in the middle instead of flowing unidirectionally from top to bottom or from bottom to top.

It should be noted that Example 2 and Comparative Example 2 use the same three-dimensional fluid focusing structure as mentioned above to prepare liposomes; the purpose is to compare high-frequency intermittent delivery of oil phase and external water phase with continuous delivery while controlling a single variable. Advantages and disadvantages of oil phase and external water phase. In fact, when the existing technology uses microfluidic technology to prepare liposomes, most of the first microchannels, the first channel 5, and the second channel 13 are processed on the same plane; the first microchannel and the first channel 5 are processed first. After the flow is merged, it is then merged with the second channel 13 to realize the flow of the internal water phase, the oil phase, and the external water phase; liposomes are prepared in a two-dimensional fluid focusing manner. In comparison, the preparation throughput and preparation rate of the three-dimensional fluid focusing used in Example 2 and Comparative Example 2 will be greatly improved. In other words, even Comparative Example 2 has the advantage of greatly improving the preparation throughput and preparation rate compared with the existing technology.

In addition, as shown in FIGS. 11 to 16 , in the first and second embodiments, the oil phase pump 4 for intermittently transporting the oil phase may adopt the following structure. The oil phase pump 4 includes: a pump chamber 16 with a variable volume chamber, a liquid inlet chamber 17 and a liquid outlet chamber 18 connected to the variable volume chamber, a liquid inlet control 19 limited to the liquid inlet chamber 17, and a liquid inlet control 19 limited to the liquid inlet chamber 17. The liquid outlet control 20 in the liquid outlet chamber 18 and the variable volume mechanism periodically change the volume of the variable volume cavity so that the liquid inlet control 19 and the liquid outlet control 20 operate periodically. By reducing the volume of the variable volume cavity and increasing the hydraulic pressure through the volume changing mechanism, the liquid inlet control 19 and the liquid outlet control 20 can each move along the liquid inlet cavity channel 17 and the liquid outlet cavity channel 18 toward the end away from the volume change cavity. When the liquid inlet control 19 remains at the end of the liquid inlet cavity 17 away from the variable volume chamber, the liquid inlet cavity 17 is disconnected; and the liquid outlet control 20 leaves the end of the liquid outlet cavity 18 close to the variable volume cavity. Move in the cavity 18 or stay at the end of the liquid outlet cavity 18 away from the variable volume cavity, and the liquid outlet cavity 18 is in a connected state. At this time, the volume of the volume changing chamber continues to decrease and the hydraulic pressure continues to increase through the volume changing mechanism; that is, the liquid in the volume changing chamber flows into the drain pipe 21 through the liquid outlet channel 18 . On the contrary, by increasing the volume of the variable volume cavity and reducing the hydraulic pressure through the variable volume mechanism, the liquid inlet control 19 and the liquid outlet control 20 can be moved toward the end of the variable volume cavity along the liquid inlet cavity channel 17 and the liquid outlet cavity channel 18 respectively. move. When the liquid outlet control 20 remains at the end of the liquid outlet channel close to the variable volume chamber, the liquid outlet cavity 18 is disconnected; and the liquid inlet control 19 leaves the end of the liquid inlet cavity 17 away from the variable volume cavity. 17 moves in, or remains at one end of the liquid inlet chamber 17 close to the variable volume chamber, and the liquid inlet chamber 17 is in a connected state; at this time, the volume of the variable volume chamber continues to increase and the hydraulic pressure continues to decrease through the volume changing mechanism; that is, The variable volume cavity can be made to suck liquid through the liquid inlet channel. There are two pump chambers 16, liquid inlet chambers 17, liquid outlet chambers 18, liquid inlet controls 19, and liquid outlet controls 20 respectively, forming two groups of liquid delivery systems. The two liquid outlet chambers 18 merge and communicate with the drain pipe 21; the volume change cycles of the volume change chambers of the two groups of liquid delivery systems are opposite. When the volume of the variable volume chamber of one group of liquid delivery systems is reduced and its liquid outlet control 20 moves along the liquid outlet channel 18 toward the end away from the variable volume chamber; the volume of the variable volume chamber of the other group of liquid delivery systems increases. If the liquid outlet control 20 is large, the liquid outlet control 20 will move along the liquid outlet channel 18 towards the end close to the variable volume chamber, and the liquid in the liquid outlet cavity 18 will flow back; at this time, one of the liquid outlet channels of one group of liquid delivery systems The liquid 18 sent to the drain pipe 21 will be compensated to the liquid outlet cavity 18 of another set of liquid delivery systems, and the liquid in the drain pipe 21 will stop flowing out. When the volume of the variable volume cavity of one group of liquid delivery systems is reduced and its liquid outlet control 20 is maintained at the end of the liquid outlet channel 18 away from the variable volume cavity; the volume of the variable volume cavity of the other group of liquid delivery systems increases. , the liquid outlet control 20 will remain at one end of the liquid outlet cavity 18 close to the variable volume chamber, so that the liquid outlet cavity 18 remains disconnected, and the liquid in the liquid outlet cavity 18 stops flowing back; at this time, one of the The liquid sent from the liquid outlet cavity 18 of the liquid delivery system to the drain pipe 21 will be smoothly discharged through the drain pipe 21, and the liquid in the drain pipe 21 will be in an outflow state. In this way, by rapidly changing the volume of the variable volume chambers of the two sets of liquid delivery systems periodically, the delivered oil phase can be quickly switched between the outflow state and the stopped outflow state.

Further, the pump chamber 16 has an open opening; the variable capacity mechanism includes: a diaphragm 22 that seals the open opening, and a power source 23 that drives the diaphragm 22 to periodically extend toward both sides; wherein the diaphragm 22 and the pump The space between the chambers 16 constitutes a variable volume chamber; a pump cover 24 is installed at the opening of the pump chamber 16, and the periphery of the diaphragm 22 is clamped between the pump chamber 16 and the pump cover 24. The pump cover 24 is provided with a recess on a side facing the diaphragm 22 to leave space for the diaphragm 22 to extend toward the pump cover 24 . When the diaphragm 22 stretches toward the pump chamber 16, the volume of the variable volume chamber decreases; when the diaphragm 22 stretches toward the pump cover 24, the volume of the variable volume chamber increases.

Further, the power source 23 is a servo motor and is connected to the diaphragm 22 through a transmission mechanism; the transmission mechanism includes: two limiting pieces 25 located on both sides of the diaphragm 22, fixedly connected to the two limiting pieces 25 and connected to the diaphragm 22 A vertical plug rod 26, an eccentric shaft 27 driven by a servo motor, and a connecting piece 28 connecting the plug rod 26 and the eccentric shaft 27; wherein, the eccentric shaft 27 includes a main shaft portion 29 and an eccentric portion 30; the connecting piece 28 One end is rotatably connected to the plug rod 26, and the other end is rotatably connected to the eccentric portion 30 of the eccentric shaft 27; the pump cover 24 forms a limiter for the plug rod 26, so that the plug rod 26 is limited to move along the length direction. It should be understood that by driving the above-mentioned transmission mechanism with a servo motor to periodically extend the diaphragm 22 on both sides, not only the period can be accurately controlled, but also the diaphragm 22 can have a higher upper frequency limit when performing periodic action.

Furthermore, the liquid inlet control 19 and the liquid outlet control 20 are both spherical in shape. The liquid inlet control 19 and the liquid outlet control 20 are partially or entirely made of iron; the liquid inlet cavity 17 is provided with a first electromagnet on the outer wall for controlling the initial position of the liquid inlet control 19; the outlet A second electromagnet for controlling the initial position of the liquid outlet control 20 is provided at a location on the outer wall of the liquid chamber channel 18 . It should be understood that the liquid inlet chamber 17 and the liquid outlet chamber 18 can be made of non-ferrous materials. Before starting the servo motor, energize the first electromagnet and the second electromagnet to attract the liquid inlet control 19 and the liquid outlet control 20; then the initial positions of the liquid inlet control 19 and the liquid outlet control 20 can be adjusted. Then close the first electromagnet and the second electromagnet, and start the servo motor, so that the liquid inlet control 19 and the liquid outlet control 20 can be in a more precise position when making periodic actions. Usually, the initial position of the liquid inlet control 19 and the liquid outlet control 20 of one group of liquid delivery systems is at one end close to the variable volume chamber, and the initial position of the liquid inlet control 19 and liquid outlet control 20 of the other group of liquid delivery systems is at the far end. One end of the volume change cavity.

Further, the inner diameter of the ports at both ends of the liquid inlet cavity 17 is smaller than the diameter of the liquid inlet control 19; the liquid inlet cavity 17 includes a first closed section 31 and a first flow section 32 butt-jointed; the first closed section The inner diameter of 31 is less than or equal to the diameter of the liquid inlet control 19; the first flow section 32 has a first main channel 33 that matches the diameter of the liquid inlet control 19, and a first auxiliary channel 34 for liquid to pass through. It should be understood that the inner diameter of the first main channel 33 is the same as or slightly larger than the diameter of the liquid inlet control 19 . The liquid inlet ports of the liquid inlet chambers 17 of the two groups of liquid delivery systems can be connected to the same liquid inlet pipe. In addition, a first connection part with a first through hole may be provided on the outer wall of the first closing section 31, a second connection part with a second through hole may be provided on the outer wall of the first flow section 32, and a second connection part with a second through hole at the end of the liquid inlet pipe. A third connecting portion with a third through hole is provided on the outer wall, and a corresponding first threaded hole is provided on the outer wall of the pump chamber 16; in this way, by passing through bolts, the liquid inlet pipe, the first closed section 31, and The first flow section 32 and the pump chamber 16.

Further, the inner diameter of the ports at both ends of the liquid outlet cavity 18 is smaller than the diameter of the liquid outlet control 20; the liquid outlet cavity 18 includes a second closed section 35 and a second flow section 36 butt-jointed; the second closed section The inner diameter of 35 is less than or equal to the diameter of the liquid outlet control 20; the second flow section 36 has a second main channel that matches the diameter of the liquid outlet control 20, and a second secondary channel for liquid to pass through. It should be understood that the inner diameter of the second main channel is the same as or slightly larger than the diameter of the liquid outlet control 20 . The structural dimensions of the liquid outlet cavity 18 and the liquid inlet cavity 17 can be set to be exactly the same; during installation, the first flow section 32 of the liquid inlet cavity 17 and the second closed section 35 of the liquid outlet cavity 18 should be oriented differently. Just accommodate the cavity. In addition, the drain pipe 21 includes an arc section and a straight section connected to the middle of the arc section, and both ends of the arc section are connected to the two liquid outlet channels 18 respectively. A fourth connection part with a fourth through hole may be provided on the outer wall of the second closing section 35 , a fifth connection part with a fifth through hole may be provided on the outer wall of the second flow section 36 , and a third connection part with a fifth through hole may be provided on the outer wall at both ends of the arc section. The sixth connection part of the six through holes is provided with a corresponding second threaded hole on the outer wall of the pump chamber 16; in this way, by passing through bolts, the drain pipe 21, the second closed section 35, and the second circulation section can be connected at once. Section 36, pump chamber 16.

In addition, both the inner water phase pump 9 and the outer water phase pump 7 can adopt the same structural principle as the oil phase pump 4 . Of course, the oil phase pump 4, the internal water phase pump 9, and the external water phase pump 7 can also adopt other structures to achieve high-frequency intermittent transportation of the internal water phase, oil phase, and external water phase.

Although specific embodiments of the present invention have been described above, those skilled in the art will understand that various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. However, these changes and Modifications all fall within the protection scope of the present invention.

Claims (10)

1. The preparation method of the liposome is characterized by comprising the following steps:

s1: dissolving lipid material and lipid-soluble material to be encapsulated in organic solvent to obtain oil phase; dissolving stabilizer in water to obtain external water phase;

s2: flowing the external aqueous phase along a first channel (5); flowing the oil phase along the first microchannel and vertically into the first channel (5) from the outlet end of the first microchannel; allowing the oil phase to diffuse into the outer aqueous phase to form liposomes;

wherein,

the first channel (5) is arranged on the first confluence piece (6), and the first channel (5) penetrates through the first confluence piece (6) along the front-back direction; the first micro-channel is arranged on the first micro-tube (1), the first micro-tube (1) is limited in the first micro-tube chamber (2), and the first micro-tube chamber (2) is positioned at the left side of the first channel (5); the first confluence piece (6) is provided with a first right channel (8) penetrating rightward from the first channel (5);

the oil phase and the external water phase are respectively and rapidly switched between a flowing state and a flowing stopping state;

the oil phase is in a flowing state, and overflows from the outlet end of the first micro-channel; the external water phase is in a stop flow state;

when the oil phase is in a stop flow state; the outer water phase is in a flowing state and is wrapped by the overflowed oil phase to flow along the first right channel (8).

2. The method for preparing the liposome according to claim 1, wherein:

the lipid substance is one or more of phospholipid, cholesterol and amphiprotic substances; the organic solvent is diethyl ether, ethanol, cyclohexane or chloroform; the stabilizer is one or more of monosaccharide, oligosaccharide, acid and alkali.

3. The method for preparing a liposome according to claim 1 or 2, characterized in that:

the first microtube (1) is provided with a plurality of first micro channels which are arranged in parallel;

the first microtube chamber (2) is provided with a plurality of first limiting through holes (3) for accommodating the first microtubes (1);

the first micropipe chamber (2) is communicated with an oil phase pump (4) for intermittently conveying the oil phase.

4. A method of preparing a liposome according to claim 3, wherein:

two ends of the first channel (5) are respectively communicated with an external water phase pump (7) for intermittently conveying external water phase;

the first microtube chamber (2) is communicated with the first channel (5) through a first microtube (1) limited in the first limiting through hole (3);

the first microtubes (1) are distributed on the front side and the rear side of the first right channel (8);

the left-right width of the first channel (5) is less than or equal to 1mm.

5. The preparation method of the liposome is characterized by comprising the following steps:

s1: dissolving lipid material in organic solvent, or dissolving lipid material and lipid-soluble material to be encapsulated in organic solvent to obtain oil phase; dissolving the water-soluble substance to be encapsulated in water to prepare an inner water phase; dissolving stabilizer in water to obtain external water phase;

s2: flowing the oil phase along a first channel (5); flowing the inner aqueous phase along the first microchannel and vertically into the first channel (5) from the outlet end of the first microchannel; preparing a mixed phase in which the water bubbles are diffused;

the first channel (5) is arranged on the first confluence piece (6), and the first channel (5) penetrates through the first confluence piece (6) along the front-back direction; the first micro-channel is arranged on the first micro-tube (1), the first micro-tube (1) is limited in the first micro-tube chamber (2), and the first micro-tube chamber (2) is positioned at the left side of the first channel (5); the first confluence piece (6) is provided with a first right channel (8) penetrating rightward from the first channel (5);

s3: flowing the external aqueous phase along a second channel (13); allowing the mixed phase to enter the second micro-channel along the first right channel (8) and vertically enter the second channel (13) from the outlet end of the second micro-channel; allowing the mixed phase to diffuse into the outer aqueous phase to form liposomes;

wherein,

the inner water phase, the oil phase and the outer water phase are respectively and rapidly switched between a flowing state and a flowing stopping state;

when the inner water phase is in a flowing state, the oil phase and the outer water phase are in a stop flowing state;

when the oil phase is in a flowing state, the inner water phase and the outer water phase are in a stop flowing state;

when the outer water phase is in a flowing state, the inner water phase and the oil phase are in a stop flowing state.

6. The method for producing a liposome according to claim 5, wherein:

the internal aqueous phase is dissolved with a stabilizer; the stabilizer is one or more of monosaccharide, oligosaccharide, acid and alkali; the lipid substance is one or more of phospholipid, cholesterol and amphiprotic substances; the organic solvent is diethyl ether, ethanol, cyclohexane or chloroform.

7. The method for preparing a liposome according to claim 5 or 6, wherein:

the first microtube (1) is provided with a plurality of first micro channels which are arranged in parallel;

the first microtube chamber (2) is provided with a plurality of first limiting through holes (3) for accommodating the first microtubes (1);

the first microtube chamber (2) is communicated with an internal water phase pump (9) for intermittently conveying the internal water phase.

8. The method for preparing a liposome according to claim 7, wherein:

two ends of the first channel (5) are respectively communicated with an oil phase pump (4) for intermittently conveying the oil phase;

the first microtube chamber (2) is communicated with the first channel (5) through a first microtube (1) limited in the first limiting through hole (3);

the first microtubes (1) are distributed on the front side and the rear side of the first right channel (8); the left-right width of the first channel (5) is less than or equal to 1mm.

9. The method for preparing the liposome according to claim 8, wherein:

the second micro-channel is arranged on the second micro-tube (10), and the second micro-tube (10) is provided with a plurality of second micro-channels which are arranged in parallel;

the second microtube (10) is limited in a second microtube chamber (11), and the second microtube chamber (11) is provided with a plurality of second limiting through holes (12) for accommodating the second microtube (10); the second microtube chamber (11) is in communication with the first right channel (8).

10. The method for preparing the liposome according to claim 9, wherein:

the second channel (13) is arranged on the second confluence piece (14); the second channel (13) penetrates through the second confluence piece (14) along the up-down direction, and two ends of the second channel are respectively communicated with an external water phase pump (7) for intermittently conveying external water phase;

the second microtube chamber (11) is positioned at the left side of the second channel (13), and the second microtube chamber are communicated through a second microtube (10) limited in a second limiting through hole (12);

the second confluence piece (14) is provided with a second right channel (15) penetrating rightward from the second channel (13); the second microtubes (10) are distributed on the upper side and the lower side of the second right channel (15); the width of the second channel (13) is less than or equal to 1mm.