CN114887562A - Method for preparing liposome - Google Patents

Method for preparing liposome Download PDF

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Publication number
CN114887562A
CN114887562A CN202210531865.5A CN202210531865A CN114887562A CN 114887562 A CN114887562 A CN 114887562A CN 202210531865 A CN202210531865 A CN 202210531865A CN 114887562 A CN114887562 A CN 114887562A
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channel
micro
phase
water phase
oil phase
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CN114887562B (en
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孙毅毅
羊向新
谢来宾
陈梨花
甘红星
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Jiangxi Chundi Biotechnology Co ltd
Chengdu Kejian Biomedical Co ltd
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Jiangxi Chundi Biotechnology Co ltd
Chengdu Kejian Biomedical Co ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J14/00—Chemical processes in general for reacting liquids with liquids; Apparatus specially adapted therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements

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  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
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Abstract

The invention provides a preparation method of liposome, aiming at solving the technical problem of low preparation speed of the existing liposome preparation method. The adopted technical scheme is as follows: the preparation method of the liposome comprises the following steps: s1: dissolving lipid substances and fat-soluble substances to be encapsulated in an organic solvent to prepare an oil phase; dissolving the stabilizer in water to prepare an external water phase; s2: flowing the external aqueous phase along the first channel; enabling the oil phase to flow along the first microchannel and vertically enter the first channel from the outlet end of the first microchannel; the oil phase is allowed to diffuse into the external aqueous phase, forming liposomes. Wherein, the oil phase and the external water phase are rapidly and alternately switched between a flowing state and a flowing stopping state respectively. The invention can break the flow rate limitation of the oil phase and improve the preparation speed of the liposome.

Description

Method for preparing liposome
Technical Field
The invention relates to the technical field of liposome production, in particular to a preparation method of liposome.
Background
When amphiphilic molecules such as phospholipids and sphingolipids are dispersed in the internal aqueous phase, the hydrophobic tails of the molecules tend to cluster together, avoiding the internal aqueous phase, while the hydrophilic heads are exposed to the internal aqueous phase, forming closed vesicles with a bilayer structure, known as liposomes. The liposome is used as a carrier and has wide application in the fields of cosmetics, skin care products, clinical medicine and the like.
The application of microfluidic technology to prepare liposomes is a current research hotspot. A common method for preparing liposome by micro-fluidic technology is to make the oil phase dissolved with the substance to be encapsulated flow along the micro-channel and make the external water phase flow through the outlet end of the micro-channel, so as to extrude and wrap the oil phase flowing out of the micro-channel, and make the oil phase disperse in the external water phase to form liposome.
In the prior art, micro-injection pumps are mostly adopted to provide power for the oil phase and the external water phase, so that the oil phase and the external water phase are crossed at a basically constant speed. When the method is adopted to prepare the liposome; in order to allow the oil phase to disperse well into the external aqueous phase to form liposomes, rather than forming a stratified fluid in parallel with the external aqueous phase; the flow rate of the oil phase needs to be limited very slowly, and the defects of low preparation flux and slow preparation speed exist.
Disclosure of Invention
The invention aims to provide a preparation method of liposome. It rapidly switches the oil phase between a flow state and a stopped flow state, intermittently diffusing into the external aqueous phase to form liposomes. Therefore, parallel layered fluid can be effectively prevented from being formed with the external water phase when the oil phase flows rapidly, the flow rate limitation of the oil phase is broken, and the preparation flux and the preparation speed of the liposome are improved.
In particular, the amount of the solvent to be used,
the preparation method of the liposome comprises the following steps:
s1: dissolving lipid substances and fat-soluble substances to be encapsulated in an organic solvent to prepare an oil phase; dissolving the stabilizer in water to prepare an external water phase;
s2: flowing the external aqueous phase along the first channel; enabling the oil phase to flow along the first microchannel and vertically enter the first channel from the outlet end of the first microchannel; allowing the oil phase to diffuse into the external aqueous phase to form liposomes;
wherein the oil phase and the external water phase are rapidly switched between a flowing state and a stopped flowing state respectively; the oil phase is in a flowing state, and when the oil phase overflows from the outlet end of the first microchannel, the external water phase is in a stopped flowing state; when the oil phase is in a stop flowing state, the external water phase is in a flowing state and wraps the overflowed oil phase to flow along the first channel.
The working principle of the invention is as follows: enabling the oil phase to flow along the first microchannel and vertically enter the first channel from the outlet end of the first microchannel; the outer aqueous phase is caused to flow along the first channel. The oil phase and the external water phase are alternately switched between a flowing state and a stopping flowing state. When the oil phase is in a flowing state; the outer aqueous phase is in a stopped flow state so that the oil phase more easily overflows from the outlet end of the first microchannel. When the oil phase is in a stopped flow state; the outer water phase is in a flowing state and can flow along the first channel with the overflowed oil phase; thereby dispersing the oil phase in the external water phase to form the liposome.
Therefore, the beneficial effects of the invention are as follows: the oil phase overflows intermittently and diffuses into the external water phase to form liposome; can effectively avoid the parallel layered fluid formed by the oil phase and the external water phase when the oil phase flows rapidly, breaks the flow rate limitation of the oil phase, and is beneficial to improving the preparation flux and the preparation speed of the liposome.
Optionally, the lipid substance is one or more of phospholipid, cholesterol and amphoteric substance; the organic solvent is diethyl ether, ethanol, cyclohexane or chloroform; the stabilizer is one or more of monosaccharide, oligosaccharide, acid and alkali.
Optionally, the first microchannel is disposed in a first microtube, and the first microtube has a plurality of first microchannels arranged in parallel; the first microtube is limited in a first microtube chamber, and the first microtube chamber is provided with a plurality of first limiting through holes for accommodating the first microtube; the first micro-tube chamber is communicated with an oil phase pump which discontinuously conveys an oil phase.
Optionally, the first channel is disposed in the first bus bar; the first channel penetrates through the first confluence piece along the front-back direction, and two ends of the first channel are respectively communicated with an external water phase pump for discontinuously conveying an external water phase; the first micro-tube chamber is positioned on the left side of the first channel and communicated with the first micro-tube chamber through a first micro-tube limited in the first limiting through hole; the first confluence piece is provided with a first right channel penetrating from the first channel to the right; the micro-tubes are distributed on the front side and the rear side of the first right channel; the left width and the right width of the first channel are less than or equal to 1 mm.
The invention also provides another preparation method of the liposome, which comprises the following steps:
s1: dissolving lipid substance in organic solvent, or dissolving lipid substance and liposoluble substance to be encapsulated in organic solvent to obtain oil phase; dissolving the water-soluble substance to be encapsulated in water to prepare an internal water phase; dissolving the stabilizer in water to prepare an external water phase;
s2: flowing the oil phase along the first channel; enabling the internal aqueous phase to flow along the first microchannel and vertically enter the first microchannel from the outlet end of the first microchannel; preparing a mixed phase with water bubbles diffused;
s3: allowing the external aqueous phase to flow along the second channel; enabling the mixed phase to enter a second microchannel along the first channel and vertically enter the second channel from the outlet end of the second microchannel; allowing the mixed phase to diffuse into the external 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 stopped flowing state; when the oil phase is in a flowing state, the inner water phase and the outer water phase are in a stopped flowing state; when the external water phase is in a flowing state, the internal water phase and the oil phase are in a stopped flowing state.
Optionally, a stabilizer is dissolved in the inner water phase; the stabilizer is one or more of monosaccharide, oligosaccharide, acid and alkali; the lipid substance is one or more of phospholipid, cholesterol and amphoteric substance; the organic solvent is diethyl ether, ethanol, cyclohexane or chloroform.
Optionally, the first microchannel is disposed in a first microtube, and the first microtube has a plurality of first microchannels arranged in parallel; the first microtube is limited in a first microtube chamber, and the first microtube chamber is provided with a plurality of first limiting through holes for accommodating the first microtube; the first micro-tube chamber is communicated with an internal water phase pump which discontinuously conveys the internal water phase.
Optionally, the first channel is disposed in the first bus bar; the first channel penetrates through the first confluence piece along the front-back direction, and two ends of the first channel are respectively communicated with an oil phase pump for discontinuously conveying an oil phase; the first micro-tube chamber is positioned on the left side of the first channel and communicated with the first micro-tube chamber through a first micro-tube limited in the first limiting through hole; the first confluence piece is provided with a first right channel penetrating from the first channel to the right; the micro-tubes are distributed on the front side and the rear side of the first right channel; the left width and the right width of the first channel are less than or equal to 1 mm.
Optionally, the second microchannel is disposed in a second microtube, and the second microtube has a plurality of second microchannels arranged in parallel; the second microtube is limited in a second microtube chamber, and the second microtube chamber is provided with a plurality of second limiting through holes for accommodating the second microtube; the second micro-tube chamber is in communication with the first right channel.
Optionally, the second channel is disposed in the second bus bar; the second channel penetrates through the second confluence piece along the up-down direction, and two ends of the second channel are respectively communicated with an external water phase pump for discontinuously conveying an external water phase; the second micro-tube chamber is positioned on the left side of the second channel and communicated with the second micro-tube chamber through a second micro-tube limited in a second limiting through hole; the second confluence piece is provided with a second right channel penetrating through the second channel rightwards; the second micro-tubes are distributed on the upper side and the lower side of the second right channel; the left width and the right width of the second channel are less than or equal to 1 mm.
The working principle of the invention is as follows: firstly, driving an inner water phase to flow along a first micro-channel by an inner water phase pump, and stopping the flow of an oil phase and an outer water phase; then, the external water phase pump drives the external water phase to flow along the second channel, and simultaneously, the internal water phase and the oil phase stop flowing; then the oil phase pump drives the oil phase to flow along the first channel, and simultaneously the inner water phase and the outer water phase stop flowing; finally, the external water phase pump drives the external water phase to flow along the second channel, and the internal water phase and the oil phase stop flowing at the same time; thus, the liposome can be obtained by repeating the above steps. 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 also overflows from the outlet end of the second microchannel; and then the external water phase pump drives the external water phase to flow along the second channel, so that the external water can be extruded and wrapped relative to the overflowing mixed phase, and the mixed phase is dispersed into the external water phase to form the liposome. When the oil phase pump drives the oil phase to flow along the first channel, the oil phase is extruded and wrapped relative to the overflowed inner water phase, and the inner water phase is dispersed into the oil phase to form water bubbles; meanwhile, the mixed phase can overflow from the outlet end of the second microchannel; and then the external water phase pump drives the external water phase to flow along the second channel, so that the external water can be extruded and wrapped relative to the overflowing mixed phase, and the mixed phase is dispersed into the external water phase to form the liposome.
Therefore, the beneficial effects of the invention are as follows: the inner water phase is intermittently overflowed and dispersed into the oil phase to form a mixed phase with water bubbles dispersed; the mixed phase is intermittently overflowed and dispersed in the external aqueous phase to form liposomes. Not only the parallel layered fluid formed by the inner water phase and the oil phase when the inner water phase flows fast is avoided, but also the parallel layered fluid formed by the mixed phase and the outer water phase when the mixed phase flows fast is avoided; breaks through the flow rate limitation of the internal water phase and the oil phase, and is beneficial to improving the preparation flux and the preparation speed of the liposome.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a plot of the preparation rate versus encapsulation efficiency coordinates for example one and comparative example one;
FIG. 2 is a plot of the preparation rate versus encapsulation rate coordinates for example two and comparative example two;
FIG. 3 is a schematic diagram of the combination of the oil phase pump, the external water phase pump, and the first confluence member;
FIG. 4 is a schematic view of a first bus bar;
FIG. 5 is a schematic view of the assembly of the first bus bar and the first micro tube;
FIG. 6 is a schematic structural diagram of a first microtube;
FIG. 7 is a schematic view showing the combination of the internal water phase pump, the oil phase pump, the external water phase pump, the first confluence member and the second confluence member;
FIG. 8 is a schematic view of the connection of the first bus bar to the second bus bar;
FIG. 9 is a schematic view of the assembly of the first bus bar and the second bus bar;
FIG. 10 is a schematic view of another angle of FIG. 9;
FIG. 11 is a schematic view of the structure of an oil phase pump;
FIG. 12 is a schematic view of the assembly of the transmission;
FIG. 13 is a schematic view of the linkage mechanism coupled to the diaphragm;
FIG. 14 is an assembled view of the pump chamber, the liquid inlet channel and the liquid outlet channel;
FIG. 15 is a schematic view of the assembly of the inlet channel;
FIG. 16 is a schematic view of another angle of FIG. 15;
reference numerals: 1. a first microtube; 2. a first microtube chamber; 3. a first limiting through hole; 4. an oil phase pump; 5. a first channel; 6. a first bus bar; 7. an external water phase pump; 8. a first right channel; 9. an internal water phase pump; 10. a second microtube; 11. a second microtube chamber; 12. a second limiting through hole; 13. a second channel; 14. a second bus bar; 15. a second right channel; 16. a pump chamber; 17. a liquid inlet channel; 18. a liquid outlet channel; 19. a liquid inlet control; 20. a liquid outlet control piece; 21. a liquid discharge pipe; 22. a diaphragm; 23. a power source; 24. a pump cover; 25. a limiting member; 26. a stopper rod; 27. an eccentric shaft; 28. a connecting member; 29. a main shaft portion; 30. an eccentric portion; 31. a first closing section; 32. a first flow-through section; 33. a first main channel; 34. a first secondary channel; 35. a second closing section; 36. a second flow-through section.
Detailed Description
In the following, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships illustrated in fig. 3 or 7 for convenience in describing and simplifying the present invention, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and therefore should not be considered limiting of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; the connection can be mechanical connection, electrical connection or communication; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Example one
S1: dissolving vitamin A, phospholipid and cholesterol in chloroform to obtain oil phase with vitamin A concentration of 10g/L, phospholipid concentration of 30g/L and cholesterol concentration of 20 g/L; dissolving phosphoric acid and glucose in water to obtain external water phase with pH of 7.4 and sugar concentration of 1000 mmol/L.
S2: the oil phase pump 4 is first operated to drive the oil phase to flow along the first microchannel, while the external aqueous phase pump 7 is stopped to drive the external aqueous phase to flow.
S3: then, the external water phase pump 7 drives the external water phase to flow along the first passage 5, while the oil phase pump 4 stops driving the oil phase to flow.
S4: the steps S2, S3 are cycled at least once per second, and the number of cycles per second is proportional to the flow rate per second. The vitamin a liposome solution discharged from the first channel 5 is collected.
Example two
S1: dissolving phospholipid and cholesterol in anhydrous ethanol to obtain oil phase with phospholipid concentration of 50g/L and cholesterol concentration of 20 g/L; dissolving hydrochloric acid and adriamycin in water to prepare an internal water phase with pH of 4 and adriamycin concentration of 10 g/L; dissolving hydrochloric acid and glucose in water to obtain external water phase with pH of 4 and sugar concentration of 1200 mmol/L.
S2: firstly, an internal water phase pump 9 drives an internal water phase to flow along a first micro-channel; at the same time, the oil phase pump 4 stops driving the oil phase to flow, and the external water phase pump 7 stops driving the external water phase to 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 to flow, and the oil phase pump 4 stops driving the oil phase to flow.
S4: then the oil phase pump 4 drives the oil phase to flow along the first channel 5; at the same time, the inner water phase pump 9 stops driving the inner water phase to flow, and the outer water phase pump 7 stops driving the outer water phase to 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 to flow, and the oil phase pump 4 stops driving the oil phase to flow.
S6: the steps S2-S5 are cycled at least once per second, and the number of cycles per second is proportional to the flow rate per second. The doxorubicin liposome solution discharged from the second channel 13 was collected.
Comparative example 1
S1: dissolving vitamin A, phospholipid and cholesterol in chloroform to obtain oil phase with vitamin A concentration of 10g/L, phospholipid concentration of 30g/L and cholesterol concentration of 20 g/L; dissolving phosphoric acid and glucose in water to obtain external water phase with pH of 7.4 and sugar concentration of 1000 mmol/L.
S2: the oil phase is injected into the first micro-channel at a constant speed through an oil phase micro-injection pump, and the external water phase is injected into the first channel 5 at a constant speed through an external water phase micro-injection pump. The vitamin a liposome solution discharged from the first channel 5 is collected.
Comparative example No. two
S1: dissolving phospholipid and cholesterol in anhydrous ethanol to obtain oil phase with phospholipid concentration of 50g/L and cholesterol concentration of 20 g/L; dissolving hydrochloric acid and adriamycin in water to prepare an internal water phase with pH of 4 and adriamycin concentration of 10 g/L; dissolving hydrochloric acid and glucose in water to obtain external water phase with pH of 4 and sugar concentration of 1200 mmol/L.
S2: the inner water phase is injected into the first micro-channel at a constant speed through an inner water phase micro-injection pump, the oil phase is injected into the first channel 5 at a constant speed through an oil phase micro-injection pump, and the outer water phase is injected into the second channel 13 at a constant speed through an outer water phase micro-injection pump. The doxorubicin liposome solution discharged from the second channel 13 was collected.
For the first embodiment and the first comparative example, the same specifications were used for the other devices except for the oil phase pump 4 for intermittently delivering the oil phase and the micro-injection pump for uniformly delivering the oil phase, and the external water phase pump 7 for intermittently delivering the external water phase and the micro-injection pump for uniformly delivering the external water phase. The frequency of switching the conveyance state and the stopped conveyance state per unit time by the oil phase pump 4 and the external water phase pump 7 is proportional to the conveyance amount per unit time. The delivery rate of the vitamin A liposome solution prepared in the first embodiment is adjusted by adjusting the delivery amount of the oil phase pump 4 and the external water phase pump 7 in unit time; and sampling to determine the encapsulation efficiency corresponding to a different preparation rate of the example. Adjusting the delivery volume of the oil phase micro-injection pump and the external water phase micro-injection pump in unit time to adjust the rate of preparing the vitamin A liposome solution in the first comparative example; and sampling to determine the encapsulation efficiency corresponding to different preparation rates of the comparative example. The production rate was plotted on the abscissa and the encapsulation efficiency on the ordinate to prepare a graph, and the results are shown in FIG. 1.
As can be seen from fig. 1, the entrapment efficiency of the vitamin a liposome solution prepared in example one did not decrease significantly as the preparation rate increased; that is, the frequency of switching the delivery state and the delivery stop state of the oil phase pump 4 and the external water phase pump 7 in the unit time, that is, the number of times of circulating the steps S2 and S3 in the unit time, is increased; the preparation speed of the vitamin A liposome solution can be improved without obstacles. In contrast to comparative example i, the encapsulation efficiency is difficult to maintain at a good level and decreases with increasing preparation rate.
For example two and comparative example two, except for the internal water phase pump 9 for intermittently conveying the internal water phase and the internal water phase microinjection pump for uniformly conveying the internal water phase, the oil phase pump 4 for intermittently conveying the oil phase and the oil phase microinjection pump for uniformly conveying the oil phase, and the external water phase pump 7 for intermittently conveying the external water phase and the external water phase microinjection pump for uniformly conveying the external water phase; the other devices all adopt the same specification. The frequency of switching the conveyance state and the stopped conveyance state per unit time by the internal water phase pump 9, the oil phase pump 4, and the external water phase pump 7 is proportional to the conveyance amount per unit time. The delivery rate of the doxorubicin liposome solution prepared in the first example is adjusted by adjusting the delivery rate of the inner water phase pump 9, the oil phase pump 4 and the outer water phase pump 7 in unit time; and sampling to determine the encapsulation efficiency corresponding to a different preparation rate of the example. Adjusting the delivery volume of the inner water phase micro-injection pump, the oil phase micro-injection pump and the outer water phase micro-injection pump in unit time to adjust the speed of preparing the adriamycin liposome solution in the first comparative example; and sampling to determine the encapsulation efficiency corresponding to different preparation rates of the comparative example. The production rate was plotted on the abscissa and the encapsulation efficiency on the ordinate to prepare a graph, and the result is shown in FIG. 2.
As can be seen from fig. 2, the entrapment efficiency of the doxorubicin liposome solution prepared in example one did not decrease significantly with the increase of the preparation rate; that is, the frequency of switching the feeding state and the feeding-stopped state per unit time of the internal water phase pump 9, the oil phase pump 4, and the external water phase pump 7, that is, the number of times of circulating steps S2 to S5 per unit time is increased; the preparation speed of the adriamycin liposome solution can be improved without obstacles. In contrast to comparative example i, the encapsulation efficiency is difficult to maintain at a good level and decreases with increasing preparation rate.
In conclusion, the invention conveys the oil phase and the external water phase in a high-frequency alternating mode, or conveys the internal water phase, the oil phase and the external water phase in a high-frequency alternating mode; breaks through the flow rate limitation of the internal water phase and the oil phase, and is beneficial to improving the preparation flux and the preparation speed of the liposome.
As shown in fig. 3 to 6, in the first embodiment and the first comparative example, the first microchannel is disposed in a first microtube 1, and the first microtube 1 has a plurality of first microchannels arranged in parallel; the first microtube 1 is limited in a first microtube chamber 2, and the first microtube chamber 2 is provided with a plurality of first limiting through holes 3 for accommodating the first microtube 1; the first micro-tube chamber 2 is communicated with an oil phase pump 4 which intermittently conveys oil phase. It should be understood that the first microtubes 1 may be arranged in a plurality of rows along the length direction of the first channel 5, and adjacent rows are staggered. In addition, an expanded cap part can be arranged at one end of the first microtube 1 positioned in the first microtube chamber 2, and a first abutting tube plate which abuts against the cap part of the first microtube 1 is arranged in the first microtube chamber 2; and a bell mouth can be arranged at the outlet of the oil phase pump 4 to tightly press the first pressing tube plate.
Further, the first channel 5 is provided in the first bus bar 6; the first channel 5 penetrates through the first confluence piece 6 along the front-back direction, and two ends of the first channel are respectively communicated with an external water phase pump 7 for discontinuously conveying an external water phase; the first microtube chamber 2 is positioned at the left side of the first channel 5 and is communicated with the first microtube 1 limited by the first limiting through hole 3; the first confluence piece 6 is provided with a first right channel 8 which penetrates through the first channel 5 rightwards; the micro-pipes are distributed on the front side and the rear side 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 is understood that the oil phase and the external water phase are converged from the front and rear ends toward the middle first right channel 8, rather than flowing unidirectionally from front to rear or from rear to front, and the preparation throughput can be doubled.
It is noted that example one and comparative example one employ the same three-dimensional fluid focusing structure described above to prepare liposomes; the method is used for comparing the advantages and disadvantages of high-frequency intermittent oil phase and external water phase and continuous oil phase and external water phase in the case of controlling a single variable. In fact, in the prior art, when the liposome is prepared by applying the microfluidic technology, a first microchannel and a first channel 5 are mostly processed on the same plane; the first micro-channel and the first channel 5 are Y-shaped, so that the confluence of an oil phase and an external water phase is realized; liposomes are prepared in a two-dimensional fluid-focused manner. In contrast, the three-dimensional fluid focusing adopted in the first embodiment and the first comparative embodiment can greatly improve the preparation throughput and the preparation speed. That is, even in comparative example one, there is an advantage in that the production throughput and the production rate are greatly improved as compared with the prior art.
As shown in fig. 6 to 10, in the second embodiment and the second comparative embodiment, the first microchannel is disposed in the first microtube 1, and the first microtube 1 has a plurality of first microchannels arranged in parallel; the first microtube 1 is limited in a first microtube chamber 2, and the first microtube chamber 2 is provided with a plurality of first limiting through holes 3 for accommodating the first microtube 1; the first micro-tube chamber 2 is communicated with an internal water phase pump 9 which intermittently conveys the internal water phase. It should be understood that the first microtubes 1 may be arranged in a plurality of rows along the length direction of the first channel 5, and adjacent rows are staggered. In addition, an expanded cap part can be arranged at one end of the first microtube 1 positioned in the first microtube chamber 2, and a first abutting tube plate which abuts against the cap part of the first microtube 1 is arranged in the first microtube chamber 2; and a bell mouth can be arranged at the outlet of the internal water phase pump 9 to tightly press the first pressing tube plate.
Further, the first channel 5 is provided in the first bus bar 6; the first channel 5 penetrates through the first confluence piece 6 along the front-back direction, and two ends of the first channel are respectively communicated with the oil phase pump 4 for discontinuously conveying the oil phase; the first microtube chamber 2 is positioned at the left side of the first channel 5 and is communicated with the first microtube 1 limited by the first limiting through hole 3; the first confluence piece 6 is provided with a first right channel 8 which penetrates through the first channel 5 rightwards; the micro-pipes are distributed on the front side and the rear side 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 is understood that the preparation flux can be doubled by making the inner water phase and the oil phase flow in a confluence from the front and rear ends toward the middle first right channel 8, rather than flowing in a single direction from front to rear or from rear to front.
Further, the second microchannel is arranged in a second microtube 10, and the second microtube 10 is provided with a plurality of second microchannels 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 microchannel chamber 11 communicates with the first right channel 8. It should be understood that the second microtubes 10 may be arranged in a plurality of rows along the length of the second channel 13, with adjacent rows being staggered. In addition, an expanded cap may be disposed at one end of the second microtube 10 located in the second microtube chamber 11, and a second tight tube plate configured to tightly press the cap of the second microtube 10 may be disposed in the second microtube chamber 11; and a bell mouth can be arranged at the outlet of the first right channel 8 to tightly press the second pressing tube plate.
Further, the second passage 13 is provided in the second bus bar 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 the external water phase pump 7 for discontinuously conveying the external water phase; the second microtube chamber 11 is positioned at the left side of the second channel 13 and is communicated with the second microtube 10 limited by the second limit through hole 12; the second bus bar 14 has a second right passage 15 penetrating rightward from the second passage 13; the second microtubes 10 are distributed on the upper side and the lower side 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 mixed phase and the external water phase are converged from the upper and lower ends toward the middle second right channel 15, rather than flowing unidirectionally from top to bottom or from bottom to top, so that the production throughput can be doubled.
It should be noted that example two and comparative example two use the same three-dimensional fluid focusing structure described above to prepare liposomes; the method is used for comparing the advantages and disadvantages of high-frequency intermittent oil phase and external water phase and continuous oil phase and external water phase in the case of controlling a single variable. In fact, in the prior art, when the liposome is prepared by applying the microfluidic technology, a first microchannel, a first channel 5 and a second channel 13 are mostly processed on the same plane; the first microchannel converges with the first channel 5 and then converges with the second channel 13, so that the convergence of the internal water phase, the oil phase and the external water phase is realized; liposomes are prepared in a two-dimensional fluid-focused manner. Compared with the three-dimensional fluid focusing adopted by the second embodiment and the second comparative embodiment, the preparation throughput and the preparation speed are greatly improved. That is, even the comparative example has an advantage that the production throughput and the production rate are greatly improved as compared with the prior art.
As shown in fig. 11 to 16, in the first and second embodiments, the oil phase pump 4 for intermittently feeding the oil phase may have the following configuration. The oil phase pump 4 includes: the device comprises a pump chamber 16 with a variable volume cavity, a liquid inlet channel 17 and a liquid outlet channel 18 which are communicated with the variable volume cavity, a liquid inlet control 19 limited in the liquid inlet channel 17, a liquid outlet control 20 limited in the liquid outlet channel 18, and a variable volume mechanism for periodically changing the volume of the variable volume cavity to enable the liquid inlet control 19 and the liquid outlet control 20 to periodically act. The volume of the variable volume cavity is reduced and the hydraulic pressure is increased through the variable volume mechanism, so that the liquid inlet control 19 and the liquid outlet control 20 respectively move towards one end far away from the variable volume cavity along the liquid inlet channel 17 and the liquid outlet channel 18. When the liquid inlet control 19 is kept at one end of the liquid inlet channel 17 far away from the variable volume cavity, the liquid inlet channel 17 is disconnected; the liquid outlet control 20 is separated from one end of the liquid outlet channel 18 close to the variable volume cavity, and moves in the liquid outlet channel 18 or is kept at one end of the liquid outlet channel 18 far away from the variable volume cavity, and the liquid outlet channel 18 is in a communicated state. At the moment, the volume of the variable volume cavity is continuously reduced and the hydraulic pressure is continuously increased through the variable volume mechanism; the liquid in the variable volume cavity can flow into the drain pipe 21 through the liquid outlet channel 18. On the contrary, the volume of the variable volume cavity is increased and the hydraulic pressure is reduced through the variable volume mechanism, so that the liquid inlet control 19 and the liquid outlet control 20 respectively move towards one end close to the variable volume cavity along the liquid inlet channel 17 and the liquid outlet channel 18. When the liquid outlet control 20 is kept at one end of the liquid outlet channel close to the variable volume cavity, the liquid outlet channel 18 is disconnected; the liquid inlet control 19 is separated from one end of the liquid inlet channel 17 far away from the variable volume cavity, moves in the liquid inlet channel 17 or is kept at one end of the liquid inlet channel 17 close to the variable volume cavity, and the liquid inlet channel 17 is in a communicated state; at the moment, the volume of the variable volume cavity is continuously increased and the hydraulic pressure is continuously reduced through the variable volume mechanism; the liquid can be sucked into the variable volume cavity through the liquid inlet channel. Two pump chambers 16, two liquid inlet channels 17, two liquid outlet channels 18, two liquid inlet controls 19 and two liquid outlet controls 20 are arranged to form two groups of liquid conveying systems. The two liquid outlet channels 18 are communicated with a liquid discharge pipe 21 after confluence; the volume change periods of the volume-variable cavities of the two liquid conveying systems are opposite. When the volume of the variable volume cavity of one group of liquid conveying systems is reduced and the liquid outlet control 20 moves towards one end far away from the variable volume cavity along the liquid outlet channel 18; the volume of the variable volume cavity of the other group of liquid conveying systems is increased, the liquid outlet control 20 moves towards one end close to the variable volume cavity along the liquid outlet channel 18, and the liquid in the liquid outlet channel 18 flows backwards; at this time, the liquid sent from the liquid outlet channel 18 of one group of liquid sending systems to the liquid discharge pipe 21 is compensated for the liquid outlet channel 18 of the other group of liquid sending systems, and the liquid in the liquid discharge pipe 21 is in a state of stopping flowing out. When the volume of the variable volume cavity of one group of liquid conveying systems is reduced and the liquid outlet control 20 is kept at one end of the liquid outlet channel 18 far away from the variable volume cavity; the volume of the variable volume cavity of the other liquid conveying system is increased, the liquid outlet control 20 is kept at one end of the liquid outlet channel 18 close to the variable volume cavity, so that the liquid outlet channel 18 is kept in a disconnected state, and the liquid in the liquid outlet channel 18 stops flowing backwards; at this time, the liquid sent to the drain pipe 21 from the liquid outlet channel 18 of one of the liquid sending systems is smoothly discharged through the drain pipe 21, and the liquid in the drain pipe 21 flows out. In this way, the volume of the variable volume chambers of the two liquid delivery systems can be changed periodically, so that the delivered oil phase can be switched between the outflow state and the outflow stop state.
Further, the pump chamber 16 has an opening; the variable volume mechanism includes: a diaphragm 22 which is closed by the opening, and a power source 23 which drives the diaphragm 22 to periodically extend to both sides; wherein the space between the diaphragm 22 and the pump chamber 16 constitutes a positive displacement chamber; the pump chamber 16 is fitted with a pump cover 24 at the opening, and the periphery of the diaphragm 22 is clamped between the pump chamber 16 and the pump cover 24. The pump cap 24 is recessed on the side facing the diaphragm 22 to allow room for the diaphragm 22 to extend toward the pump cap 24. As the diaphragm 22 extends toward the pump chamber 16, the volume of the positive displacement chamber decreases; as the diaphragm 22 extends toward the pump cap 24, the volume of the positive displacement chamber increases.
Further, the power source 23 is a servo motor and is connected with the diaphragm 22 through a transmission mechanism; the transmission mechanism includes: two limit parts 25 positioned on two sides of the diaphragm 22, a plug rod 26 fixedly connected with the two limit parts 25 and vertical to the diaphragm 22, 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 comprises a main shaft part 29 and an eccentric part 30; one end of the connecting piece 28 is rotationally connected with the plug rod 26, and the other end is rotationally connected with the eccentric part 30 of the eccentric shaft 27; the pump cover 24 limits the plug rod 26, so that the plug rod 26 is limited to move along the length direction. It will be appreciated that the use of a servo motor to drive the actuator to periodically extend the diaphragm 22 on both sides not only allows for precise control of the cycle, but also allows for a higher upper frequency limit for the cyclic motion of the diaphragm 22.
Further, the liquid inlet control 19 and the liquid outlet control 20 are both spherical. The liquid inlet control piece 19 and the liquid outlet control piece 20 are partially or completely made of iron; a first electromagnet for controlling the initial position of the liquid inlet control piece 19 is arranged at one position of the outer wall of the liquid inlet cavity channel 17; a second electromagnet for controlling the initial position of the liquid outlet control 20 is arranged at one position of the outer wall of the liquid outlet channel 18. It should be understood that the inlet channel 17 and the outlet channel 18 may be made of non-ferrous materials. Before the servo motor is started, the first electromagnet and the second electromagnet are electrified to attract the liquid inlet control 19 and the liquid outlet control 20; the initial positions of the liquid inlet control 19 and the liquid outlet control 20 can be adjusted. Then, the first electromagnet and the second electromagnet are turned off, and the servo motor is started, so that the liquid inlet control 19 and the liquid outlet control 20 can be located at more accurate positions when the liquid inlet control and the liquid outlet control perform periodic actions. Generally, the initial positions of the liquid inlet control 19 and the liquid outlet control 20 of one group of liquid conveying systems are close to one end of the variable capacity cavity, and the initial positions of the liquid inlet control 19 and the liquid outlet control 20 of the other group of liquid conveying systems are far from one end of the variable capacity cavity.
Further, the inner diameter of the port at the two ends of the liquid inlet channel 17 is smaller than the diameter of the liquid inlet control piece 19; the liquid inlet channel 17 comprises a first closing section 31 and a first flow-through section 32 which are butted; the inner diameter of the first closing section 31 is less than or equal to the diameter of the liquid inlet control piece 19; the first flow-through section 32 has a first main channel 33 adapted to the diameter of the inlet control 19 and a first secondary channel 34 for the passage of liquid. It will be appreciated that the internal diameter of the first main channel 33 is the same as the diameter of the inlet control 19, or slightly larger than the diameter of the inlet control 19. The liquid inlet ports of the liquid inlet channels 17 of the two liquid conveying systems can be communicated with the same liquid inlet pipe. Furthermore, a first connecting part with a first through hole can be arranged on the outer wall of the first closing section 31, a second connecting part with a second through hole can be arranged on the outer wall of the first flowing section 32, a third connecting part with a third through hole can be arranged on the outer wall of the end part of the liquid inlet pipe, and a corresponding first threaded hole can be arranged on the outer wall of the pump chamber 16; thus, the liquid inlet pipe, the first closing section 31, the first circulation section 32 and the pump chamber 16 can be connected at one time by penetrating the bolts.
Further, the inner diameters of the ports at the two ends of the liquid outlet channel 18 are smaller than the diameter of the liquid outlet control 20; the liquid outlet channel 18 comprises a second closing section 35 and a second flow-through section 36 which are butted; the inner diameter of the second closing section 35 is less than or equal to the diameter of the liquid outlet control 20; the second flow-through section 36 has a second main channel adapted to the diameter of the liquid outlet control 20, and a second sub-channel for the liquid to pass through. It should be understood that the inner diameter of the second main channel is the same as the diameter of the liquid outlet control 20 or slightly larger than the diameter of the liquid outlet control 20. The structural sizes of the liquid outlet channel 18 and the liquid inlet channel 17 can be set to be completely the same; when the device is installed, the first flowing section 32 of the liquid inlet channel 17 and the second closing section 35 of the liquid outlet channel 18 are made to face the variable-volume cavity. In addition, the liquid discharge pipe 21 comprises an arc section and a straight section communicated with the middle of the arc section, and two ends of the arc section are respectively communicated with the two liquid outlet channels 18. A fourth connecting part with a fourth through hole can be arranged on the outer wall of the second closing section 35, a fifth connecting part with a fifth through hole can be arranged on the outer wall of the second circulating section 36, a sixth connecting part with a sixth through hole can be arranged on the outer walls of the two ends of the arc section, and a corresponding second threaded hole can be arranged on the outer wall of the pump chamber 16; in this way, the drain pipe 21, the second closing section 35, the second flow passage section 36, and the pump chamber 16 can be connected at one time by bolting.
In addition, the internal water phase pump 9 and the external 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 realize the high-frequency intermittent conveying of the internal water phase, the oil phase and the external water phase.
Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that changes or modifications may be made to these embodiments without departing from the principles and spirit of the invention, and that such changes and modifications are within the scope of the invention.

Claims (10)

1. The preparation method of the liposome is characterized by comprising the following steps:
s1: dissolving lipid substances and fat-soluble substances to be encapsulated in an organic solvent to prepare an oil phase; dissolving the stabilizer in water to prepare an external water phase;
s2: -allowing the external aqueous phase to flow along the first channel (5); allowing the oil phase to flow along the first microchannel and vertically enter the first channel (5) from the outlet end of the first microchannel; allowing the oil phase to diffuse into the external aqueous phase to form liposomes;
wherein the content of the first and second substances,
the oil phase and the external water phase are rapidly switched between a flowing state and a stopped flowing state respectively;
the oil phase is in a flowing state and overflows from the outlet end of the first microchannel; the external water phase is in a stop flow state;
when the oil phase is in a stop flowing state; the external water phase is in a flowing state and flows along the first channel (5) with the overflowed oil phase.
2. The method for preparing a liposome according to claim 1, wherein:
the lipid substance is one or more of phospholipid, cholesterol and amphoteric substance; 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, wherein:
the first micro-channel is arranged on a first micro-tube (1), and the first micro-tube (1) is provided with a plurality of first micro-channels which are arranged in parallel;
the first microtube (1) is limited in the first microtube chamber (2), and the first microtube chamber (2) is provided with a plurality of first limiting through holes (3) for accommodating the first microtube (1);
the first micro-tube chamber (2) is communicated with an oil phase pump (4) which discontinuously conveys oil phase.
4. A method of preparing liposomes according to claim 3 wherein:
the first channel (5) is arranged on the first confluence piece (6); the first channel (5) penetrates through the first confluence piece (6) along the front-back direction, and two ends of the first channel are respectively communicated with an external water phase pump (7) for discontinuously conveying an external water phase;
the first micro-tube chamber (2) is positioned on the left side of the first channel (5) and communicated with the first micro-tube chamber through a first micro-tube (1) limited in the first limiting through hole (3);
the first confluence piece (6) is provided with a first right channel (8) penetrating from the first channel (5) to the right; the micro-tubes are distributed on the front side and the rear side of the first right channel (8);
the left and right width of the first channel (5) is less than or equal to 1 mm.
5. The preparation method of the liposome is characterized by comprising the following steps:
s1: dissolving lipid substance in organic solvent, or dissolving lipid substance and liposoluble substance to be encapsulated in organic solvent to obtain oil phase; dissolving the water-soluble substance to be encapsulated in water to prepare an internal water phase; dissolving the stabilizer in water to prepare an external water phase;
s2: -causing the oil phase to flow along the first channel (5); allowing the internal aqueous phase to flow along the first microchannel and vertically enter the first channel (5) from the outlet end of the first microchannel; preparing a mixed phase with water bubbles diffused;
s3: allowing the external aqueous phase to flow along the second channel (13); allowing the mixed phase to enter the second microchannel along the first channel (5) and vertically enter the second channel (13) from the outlet end of the second microchannel; allowing the mixed phase to diffuse into the external aqueous phase to form liposomes;
wherein the content of the first and second substances,
the inner water phase, the oil phase and the outer water phase are rapidly switched between a flowing state and a stopped flowing state respectively;
when the inner water phase is in a flowing state, the oil phase and the outer water phase are in a stopped flowing state;
when the oil phase is in a flowing state, the inner water phase and the outer water phase are in a stopped flowing state;
when the external water phase is in a flowing state, the internal water phase and the oil phase are in a stopped flowing state.
6. The method for preparing a liposome according to claim 5, wherein:
a stabilizer is dissolved in the inner water phase; the stabilizer is one or more of monosaccharide, oligosaccharide, acid and alkali; the lipid substance is one or more of phospholipid, cholesterol and amphoteric substance; 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 micro-channel is arranged on a first micro-tube (1), and the first micro-tube (1) is provided with a plurality of first micro-channels which are arranged in parallel;
the first microtube (1) is limited in the first microtube chamber (2), and the first microtube chamber (2) is provided with a plurality of first limiting through holes (3) for accommodating the first microtube (1);
the first micro-tube chamber (2) is communicated with an internal water phase pump (9) which discontinuously conveys the internal water phase.
8. The method for preparing a liposome according to claim 7, wherein:
the first channel (5) is arranged on the first confluence piece (6); the first channel (5) penetrates through the first confluence piece (6) along the front-back direction, and two ends of the first channel are respectively communicated with the oil phase pump (4) for discontinuously conveying the oil phase;
the first micro-tube chamber (2) is positioned on the left side of the first channel (5) and communicated with the first micro-tube chamber through a first micro-tube (1) limited in the first limiting through hole (3);
the first confluence piece (6) is provided with a first right channel (8) penetrating from the first channel (5) to the right; the micro-tubes are distributed on the front side and the rear side of the first right channel (8); the left and right width of the first channel (5) is less than or equal to 1 mm.
9. The method for preparing a liposome according to claim 8, wherein:
the second micro-channel is arranged on a 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 micro-tube chamber (11) is communicated with the first right channel (8).
10. The method for preparing a liposome according to claim 9, wherein:
the second channel (13) is arranged on a second confluence piece (14); the second channel (13) penetrates through the second confluence piece (14) along the vertical direction, and two ends of the second channel are respectively communicated with an external water phase pump (7) for discontinuously conveying an external water phase;
the second micro-tube chamber (11) is positioned on the left side of the second channel (13) and communicated with the second micro-tube chamber through a second micro-tube (10) limited in a second limiting through hole (12);
the second confluence piece (14) is provided with a second right channel (15) penetrating from the second channel (13) to the right; the second micro-tubes (10) are distributed on the upper side and the lower side of the second right channel (15); the left and right width of the second channel (13) is less than or equal to 1 mm.
CN202210531865.5A 2022-05-17 2022-05-17 Preparation method of liposome Active CN114887562B (en)

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CN112120022A (en) * 2020-09-29 2020-12-25 江苏擎宇化工科技有限公司 Blank multivesicular liposome and preparation method and device thereof
CN113499697A (en) * 2021-08-04 2021-10-15 西南交通大学 Water-in-water monodisperse double emulsion and preparation method thereof
CN113600251A (en) * 2021-08-06 2021-11-05 中南大学 3D printing multilayer microfluidic chip and high-throughput liposome preparation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112184A1 (en) * 2003-11-26 2005-05-26 Andreas Jahn Controlled vesicle self-assembly in continuous two phase flow microfluidic channels
US20060220269A1 (en) * 2005-03-29 2006-10-05 Yasuko Noritomi Method for manufacturing multiple-phase particle and apparatus for manufacturing multiple-phase particle
CN1846844A (en) * 2005-03-29 2006-10-18 株式会社东芝 Method for manufacturing multiple-phase particle and apparatus for manufacturing multiple-phase particle
CN1895223A (en) * 2006-04-05 2007-01-17 沈阳药科大学 Production of elaioplast
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