CN114887562B - Preparation method of liposome - Google Patents

Preparation method of liposome Download PDF

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Publication number
CN114887562B
CN114887562B CN202210531865.5A CN202210531865A CN114887562B CN 114887562 B CN114887562 B CN 114887562B CN 202210531865 A CN202210531865 A CN 202210531865A CN 114887562 B CN114887562 B CN 114887562B
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channel
phase
water phase
oil phase
micro
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CN114887562A (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 & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Medicinal Preparation (AREA)

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 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 an internal aqueous phase, the hydrophobic tails of the molecules tend to aggregate together, bypassing 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 preparation of liposomes using microfluidic technology is currently a research hotspot. One common method for preparing liposomes by microfluidic technology is to make the oil phase in which the substance to be encapsulated is dissolved flow along the microchannel and the outer water phase flows through the outlet end of the microchannel, so as to squeeze and pinch the oil phase flowing out of the microchannel, and disperse the oil phase into the outer water phase to form the liposome.
In the prior art, a microinjection pump is mostly adopted to provide power for an oil phase and an external water phase, so that the oil phase and the external water phase meet at a basically constant speed. When the liposome is prepared by the method; in order to enable the oil phase to be well dispersed into the outer water phase to form liposome, rather than forming parallel layered fluid with the outer water phase; the flow rate of the oil phase needs to be limited to be extremely slow, and the defects of low preparation flux and slow preparation rate exist.
Disclosure of Invention
The invention aims to provide a preparation method of liposome. Which causes the oil phase to rapidly switch between a flowing state and a stopped flowing state, intermittently diffuses into the outer aqueous phase to form liposomes. Therefore, the formation of parallel layered fluid with the external water phase when the oil phase flows rapidly can be effectively avoided, the flow rate limit of the oil phase is broken, and the preparation flux and the preparation rate of the liposome are improved.
In particular, the method comprises the steps of,
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; allowing the oil phase 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 rapidly; the oil phase is in a flowing state, and when overflows from the outlet end of the first micro-channel, the outer water phase is in a flowing stopping state; when the oil phase is in a flowing stopping state, the outer water phase is in a flowing state, and the overflowed oil phase is wrapped and clamped to flow along the first right channel.
The working principle of the invention is as follows: enabling the oil phase to flow along the first micro-channel, and vertically entering the first channel from the outlet end of the first micro-channel; the external aqueous phase is caused to flow along the first channel. The oil phase and the external water phase are staggered between a flowing state and a stopped flowing state to rapidly switch. 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 wrap the overflowed oil phase to flow along the first right channel; thereby dispersing the oil phase in the outer aqueous phase to form liposomes.
Therefore, the invention has the beneficial effects that: intermittently overflowing the oil phase, and diffusing the oil phase into the outer water phase to form liposome; the method can effectively avoid the formation of parallel layered fluid with the external water phase when the oil phase flows rapidly, breaks the flow rate limit of the oil phase, and is beneficial to improving the preparation flux and the preparation rate of the liposome.
Optionally, 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.
Optionally, the first micro-channel is arranged on the first micro-tube, and the first micro-tube is provided with a plurality of first micro-channels 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 micropipe chamber is communicated with an oil phase pump for intermittently conveying the oil phase.
Optionally, the first channel is disposed on the first confluence member; 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 intermittently conveying external water phase; the first microtube chamber is positioned at the left side of the first channel, and the first microtube chamber are communicated through a first microtube limited at the first limiting through hole; the first confluence piece is provided with a first right channel penetrating rightward from the first channel; the first microtubes are distributed on the front side and the rear side of the first right channel; the left-right width of the first channel is less than or equal to 1mm.
The invention also provides another preparation method of the liposome, which comprises 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; allowing the inner water phase to flow along the first micro-channel and vertically enter the first channel from the outlet end of the first micro-channel; preparing a mixed phase in which the water bubbles are diffused;
s3: flowing the external aqueous phase along a second channel; enabling the mixed phase to enter the second micro-channel along the first right channel and vertically enter the second channel from the outlet end of the second micro-channel; allowing the mixed phase to diffuse into the outer aqueous phase to form liposomes;
the inner water phase, the oil phase and the outer water phase are respectively switched between a flowing state and a flowing stopping state rapidly; 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.
Optionally, 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.
Optionally, the first micro-channel is arranged on the first micro-tube, and the first micro-tube is provided with a plurality of first micro-channels 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 microtube chamber is communicated with an internal water phase pump for intermittently conveying an internal water phase.
Optionally, the first channel is disposed on the first confluence member; 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 intermittently conveying an oil phase; the first microtube chamber is positioned at the left side of the first channel, and the first microtube chamber are communicated through a first microtube limited at the first limiting through hole; the first confluence piece is provided with a first right channel penetrating rightward from the first channel; the first microtubes are distributed on the front side and the rear side of the first right channel; the left-right width of the first channel is less than or equal to 1mm.
Optionally, the second micro-channel is arranged on the second micro-tube, and the second micro-tube is provided with a plurality of second micro-channels 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 microtube chamber is in communication with the first right channel.
Optionally, the second channel is disposed on the second confluence member; 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 intermittently conveying the external water phase; the second microtube chamber is positioned at the left side of the second channel, and the second microtube chamber are communicated through a second microtube limited at the second limiting through hole; the second confluence piece is provided with a second right channel penetrating rightward from the second channel; the second microtubes are distributed on the upper side and the lower side of the second right channel; the left-right width of the second channel is less than or equal to 1mm.
The working principle of the invention is as follows: firstly, an inner water phase pump drives an inner water phase to flow along a first micro-channel, and meanwhile, an oil phase and an outer water phase stop flowing; then, the outer water phase pump drives the outer water phase to flow along the second channel, and simultaneously, the inner 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 outer water phase pump drives the outer water phase to flow along the second channel, and simultaneously the inner water phase and the oil phase stop flowing; thus, the above steps are repeated continuously to obtain the liposome. Specifically, when the internal water phase pump drives the internal water phase to overflow from the outlet end of the first micro-channel, the mixed phase also overflows from the outlet end of the second micro-channel; and then the outer water phase pump drives the outer water phase to flow along the second channel, so that the overflowed mixed phase of the outer water phase is extruded and wrapped, and the mixed phase is dispersed into the outer water phase to form the liposome. When the oil phase pump drives the oil phase to flow along the first channel, the oil phase can extrude and pinch the overflowed inner water phase, so that the inner water phase is dispersed into the oil phase to form water bubbles; meanwhile, the mixed phase overflows from the outlet end of the second micro-channel; and then the outer water phase pump drives the outer water phase to flow along the second channel, so that the overflowed mixed phase of the outer water phase is extruded and wrapped, and the mixed phase is dispersed into the outer water phase to form the liposome.
Therefore, the invention has the beneficial effects that: intermittently overflowing the inner water phase to disperse the inner water phase into the oil phase to form a mixed phase with dispersed water bubbles; the mixed phase overflows intermittently and disperses to the outer water phase to form liposome. Not only avoids the formation of parallel layered fluid with the oil phase when the inner water phase flows fast, but also avoids the formation of parallel layered fluid with the outer water phase when the mixed phase flows fast; breaks the flow rate limit of the inner water phase and the oil phase, and is beneficial to improving the preparation flux and the preparation rate 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 that are required in the embodiments or the description of the prior art will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of the preparation rate versus encapsulation efficiency coordinates for example one and comparative example one;
FIG. 2 is a graph of the preparation rate versus encapsulation efficiency coordinates for example two and comparative example two;
FIG. 3 is a schematic diagram of the oil phase pump, the external water phase pump, and the first manifold;
FIG. 4 is a schematic view of a first bus bar;
FIG. 5 is a schematic diagram illustrating the assembly of a first bus member and a first micro-pipe;
FIG. 6 is a schematic structural view of a first microtube;
FIG. 7 is a schematic diagram of the cooperation of an inner water phase pump, an oil phase pump, an outer water phase pump, a first confluence member, and a second confluence member;
FIG. 8 is a schematic diagram illustrating the connection of the first and second bus members;
FIG. 9 is an assembly schematic of a first bus member and a second bus member;
FIG. 10 is a schematic view of another angle of FIG. 9;
FIG. 11 is a schematic diagram of the structure of an oil phase pump;
FIG. 12 is an assembled schematic view of a transmission;
FIG. 13 is a schematic illustration of the connection of the drive mechanism to the diaphragm;
FIG. 14 is an assembled schematic view of the pump chamber, the liquid inlet channel, and the liquid outlet channel;
FIG. 15 is an assembled schematic view of the feed 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. the first limiting through hole; 4. an oil phase pump; 5. a first channel; 6. a first confluence member; 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. the second limiting through hole; 13. a second channel; 14. a second confluence member; 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; 21. a liquid discharge pipe; 22. a diaphragm; 23. a power source; 24. a pump cover; 25. a limiting piece; 26. a plug rod; 27. an eccentric shaft; 28. a connecting piece; 29. a main shaft portion; 30. a eccentric portion; 31. a first enclosed section; 32. a first flow-through section; 33. a first main channel; 34. a first sub-channel; 35. a second closing section; 36. a second circulation segment.
Detailed Description
Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways 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 should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in fig. 3 or fig. 7 are merely for convenience in describing the present invention and simplify the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
In the present invention, unless explicitly specified and limited otherwise, the terms "mounted," "connected," "secured," and the like are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally formed; the device can be mechanically connected, electrically connected and communicated; can be directly connected or indirectly connected through an intermediate medium, and can be communicated with the inside of two elements or the interaction relationship of the two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
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; phosphoric acid and glucose were dissolved in water to prepare an external aqueous phase having a pH of 7.4 and a sugar concentration of 1000 mmol/L.
S2: the oil phase pump 4 is firstly made to drive the oil phase to flow along the first micro-channel, and the outer water phase pump 7 is made to stop driving the outer water phase to flow.
S3: the external water phase pump 7 is then caused to drive the external water phase to flow along the first channel 5, while the oil phase pump 4 is caused to stop 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 per second. The vitamin a liposome solution discharged from the first right channel 8 was collected.
Example two
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 50g/L and cholesterol concentration of 20 g/L; dissolving hydrochloric acid and doxorubicin in water to obtain an inner water phase with pH of 4 and doxorubicin concentration of 10 g/L; hydrochloric acid and glucose were dissolved in water to prepare an external aqueous phase having a pH of 4 and a sugar concentration of 1200 mmol/L.
S2: the inner water phase pump 9 is firstly made to drive the inner water phase to flow along the first micro-channel; simultaneously, the oil phase pump 4 is stopped to drive the oil phase to flow, and the outer water phase pump 7 is stopped to drive the outer water phase to flow.
S3: then the external water phase pump 7 drives the external water phase to flow along the second channel 13; meanwhile, 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; meanwhile, 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 outer water phase pump 7 drives the outer water phase to flow along the second channel 13; meanwhile, 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 per second. The doxorubicin liposome solution discharged from the second right channel 15 was collected.
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; phosphoric acid and glucose were dissolved in water to prepare an external aqueous phase having a pH of 7.4 and a sugar concentration of 1000 mmol/L.
S2: the oil phase is injected into the first micro channel at a constant speed through the oil phase microinjection pump, and the outer water phase is injected into the first channel 5 at a constant speed through the outer water phase microinjection pump. The vitamin a liposome solution discharged from the first right channel 8 was collected.
Example two
S1: dissolving phospholipid and cholesterol in absolute ethanol to obtain oil phase with phospholipid concentration of 50g/L and cholesterol concentration of 20 g/L; dissolving hydrochloric acid and doxorubicin in water to obtain an inner water phase with pH of 4 and doxorubicin concentration of 10 g/L; hydrochloric acid and glucose were dissolved in water to prepare an external aqueous phase having a pH of 4 and a sugar concentration of 1200 mmol/L.
S2: the inner water phase is injected into the first micro-channel at a constant speed through the inner water phase microinjection pump, the oil phase is injected into the first channel 5 at a constant speed through the oil phase microinjection pump, and the outer water phase is injected into the second channel 13 at a constant speed through the outer water phase microinjection pump. The doxorubicin liposome solution discharged from the second right channel 15 was collected.
For the first embodiment and the first comparative embodiment, the other devices are the same in specification except for 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 outer water phase pump 7 for intermittently conveying the outer water phase and the outer water phase microinjection pump for uniformly conveying the outer water phase. The frequency of switching the conveying state and stopping the conveying state in the unit time of the oil phase pump 4 and the external water phase pump 7 is in direct proportion to the conveying amount in the unit time. The rate of preparing the vitamin A liposome solution 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 samples were taken to determine the encapsulation efficiency for each of the different preparation rates of the examples. The rate of preparing the vitamin A liposome solution in the first comparative example is adjusted by adjusting the delivery capacity of the oil phase microinjection pump and the external water phase microinjection pump in unit time; and samples were taken to measure the encapsulation efficiency corresponding to a different preparation rate of the comparative example. A graph of the coordinates was made with the preparation rate on the abscissa and the encapsulation efficiency on the ordinate, and the results are shown in fig. 1.
As can be seen from fig. 1, the encapsulation efficiency of the vitamin a liposome solution prepared in example one did not significantly decrease with increasing preparation rate; that is, as long as the frequency of switching the conveyance state and the stop conveyance state in the unit time by the oil phase pump 4, the external water phase pump 7 is increased, that is, the number of times of the circulation steps S2, S3 in the unit time; the preparation rate of the vitamin A liposome solution can be increased without barriers. In contrast, in comparative example one, the encapsulation efficiency was difficult to maintain at a good level and was continuously decreased as the preparation rate was continuously increased.
For the second embodiment and the second comparative embodiment, 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 conveying state and stopping the conveying state in the unit time of the inner water phase pump 9, the oil phase pump 4 and the outer water phase pump 7 is in direct proportion to the conveying amount in the unit time. The rate of preparing the doxorubicin liposome solution of the first embodiment is adjusted by adjusting the delivery amount per unit time of the inner water phase pump 9, the oil phase pump 4 and the outer water phase pump 7; and samples were taken to determine the encapsulation efficiency for each of the different preparation rates of the examples. The rate of preparing the doxorubicin liposome solution in the comparative example one is adjusted by adjusting the delivery rate of the inner water phase microinjection pump, the oil phase microinjection pump and the outer water phase microinjection pump in unit time; and samples were taken to measure the encapsulation efficiency corresponding to a different preparation rate of the comparative example. A graph of the coordinates was made with the preparation rate on the abscissa and the encapsulation efficiency on the ordinate, and the results are shown in fig. 2.
As can be seen from fig. 2, the encapsulation efficiency of the doxorubicin liposome solution prepared in example one did not significantly decrease with increasing preparation rate; that is, as long as the frequency of switching the conveyance state and the stop conveyance state in a unit time by the inner aqueous phase pump 9, the oil phase pump 4, and the outer aqueous phase pump 7 is increased, that is, the number of times of circulating steps S2 to S5 in a unit time; the preparation rate of the doxorubicin liposome solution can be increased without obstacle. In contrast, in comparative example one, the encapsulation efficiency was difficult to maintain at a good level and was continuously decreased as the preparation rate was continuously increased.
In summary, the invention conveys the oil phase and the outer water phase in a high-frequency alternating mode, or conveys the inner water phase, the oil phase and the outer water phase in a high-frequency alternating mode; breaks the flow rate limit of the inner water phase and the oil phase, and is beneficial to improving the preparation flux and the preparation rate of the liposome.
As shown in fig. 3 to 6, in the first embodiment and the first comparative example, the first micro-channel is provided in the first micro-tube 1, and the first micro-tube 1 has a plurality of first micro-channels 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 micropipe chamber 2 is communicated with an oil phase pump 4 for intermittently conveying the oil phase. It should be understood that the first microtubes 1 may be arranged in a plurality of columns along the length of the first channel 5, with adjacent columns being staggered. In addition, an enlarged 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 socket can be arranged at the outlet of the oil phase pump 4 to form propping for the first propping tube plate.
Further, the first channel 5 is provided on the first confluence member 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 intermittently conveying external water phase; the first microtube chamber 2 is positioned at the left side of the first channel 5, and the first microtube chamber are communicated through the first microtube 1 limited at the first limiting through hole 3; the first confluence member 6 has a first right channel 8 penetrating rightward from the first channel 5; the first microtubes 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. It will be appreciated that doubling the production throughput can be achieved by converging the oil and outer aqueous phases from the front and rear ends towards the central first right channel 8, rather than flowing unidirectionally from front to rear or back to front.
It should be noted that the same three-dimensional fluid focusing structure as described above was used for preparing liposomes in example one and comparative example one; the method is used for comparing the advantages and disadvantages of high-frequency intermittent conveying of the oil phase and the outer water phase with continuous conveying of the oil phase and the outer water phase under the condition of controlling a single variable. In fact, in the prior art, when the microfluidic technology is applied to preparing the liposome, the first micro-channel and the 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 the oil phase and the external water phase is realized; liposomes are prepared in a two-dimensional fluid focus. In contrast, the three-dimensional fluid focus used in examples one and comparative example one greatly increased the throughput and rate of preparation. That is, even in comparative example one, there is an advantage in that the preparation throughput and the preparation rate are greatly improved over the prior art.
As shown in fig. 6 to 10, in the second embodiment and the second comparative example, the first micro-channel is provided in the first micro-tube 1, and the first micro-tube 1 has a plurality of first micro-channels 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 microtube chamber 2 is communicated with an internal water phase pump 9 for intermittently conveying the internal water phase. It should be understood that the first microtubes 1 may be arranged in a plurality of columns along the length of the first channel 5, with adjacent columns being staggered. In addition, an enlarged 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 socket can be arranged at the outlet of the internal water phase pump 9 to form abutting against the first abutting tube plate.
Further, the first channel 5 is provided on the first confluence member 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 intermittently conveying the oil phase; the first microtube chamber 2 is positioned at the left side of the first channel 5, and the first microtube chamber are communicated through the first microtube 1 limited at the first limiting through hole 3; the first confluence member 6 has a first right channel 8 penetrating rightward from the first channel 5; the first microtubes 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. It will be appreciated that doubling the production throughput can be achieved by converging the inner aqueous and oil phases from the front and rear ends towards the central first right channel 8, rather than flowing unidirectionally from front to rear or back to front.
Further, 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 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 communicates with the first right channel 8. It should be understood that the second microtubes 10 may be arranged in a plurality of columns along the length of the second channel 13, with adjacent columns being staggered. In addition, an enlarged cap portion may be provided at one end of the second microtube 10 located in the second microtube chamber 11, and a second abutting tube plate for abutting against the cap portion of the second microtube 10 may be provided in the second microtube chamber 11; and a socket can be arranged at the outlet of the first right channel 8 to form abutting against the second abutting tube plate.
Further, the second channel 13 is provided on the second confluence member 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 intermittently conveying the 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 at a second limiting through hole 12; the second confluence member 14 has 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 1mm or less. It will be appreciated that doubling the production throughput can be achieved by converging the mixed phase, the outer aqueous phase, from the upper and lower ends towards the middle second right channel 15, rather than flowing unidirectionally from top to bottom or from bottom to top.
It should be noted that the same three-dimensional fluid focusing structure is used for preparing the liposome in the second embodiment and the second comparative embodiment; the method is used for comparing the advantages and disadvantages of high-frequency intermittent conveying of the oil phase and the outer water phase with continuous conveying of the oil phase and the outer water phase under the condition of controlling a single variable. In fact, in the prior art, when the microfluidic technology is applied to preparing the liposome, the first micro-channel, the first channel 5 and the second channel 13 are mostly processed on the same plane; the first micro-channel is converged with the first channel 5 and then converged with the second channel 13, so that the convergence of an inner water phase, an oil phase and an outer water phase is realized; liposomes are prepared in a two-dimensional fluid focus. In contrast, the three-dimensional fluid focus used in examples two and comparative example two greatly increases the throughput and rate of preparation. That is, even in comparative example II, there is an advantage in that the preparation throughput and the preparation 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 structure. The oil phase pump 4 includes: the variable-volume pump comprises a pump chamber 16 with a variable-volume cavity, a liquid inlet cavity channel 17 and a liquid outlet cavity channel 18 which are communicated with the variable-volume cavity, a liquid inlet control 19 limited in the liquid inlet cavity channel 17, a liquid outlet control 20 limited in the liquid outlet cavity channel 18, and a variable-volume mechanism for periodically changing the volume of the variable-volume cavity so as 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 through the variable-volume mechanism, and the hydraulic pressure is increased, so that the liquid inlet control piece 19 and the liquid outlet control piece 20 can respectively move along the liquid inlet cavity channel 17 and the liquid outlet cavity channel 18 towards one end far away from the variable-volume cavity. When the liquid inlet control 19 is kept at one end of the liquid inlet cavity channel 17 far away from the variable-volume cavity, the liquid inlet cavity channel 17 is disconnected; the liquid outlet control 20 leaves the end of the liquid outlet channel 18 close to the variable-volume cavity, moves in the liquid outlet channel 18 or is kept at the end of the liquid outlet channel 18 far away from the variable-volume cavity, and the liquid outlet channel 18 is in a communicating state. At this time, 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 chamber can flow into the liquid discharge pipe 21 through the liquid outlet channel 18. Conversely, 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 piece 19 and the liquid outlet control piece 20 can respectively move along the liquid inlet cavity channel 17 and the liquid outlet cavity channel 18 towards one end close to the variable-volume cavity. When the tapping control 20 is kept at one end of the tapping channel close to the variable-volume cavity, the tapping channel 18 is disconnected; the liquid inlet control 19 leaves one end of the liquid inlet cavity channel 17 far away from the variable-volume cavity, moves in the liquid inlet cavity channel 17 or is kept at one end of the liquid inlet cavity channel 17 near the variable-volume cavity, and the liquid inlet cavity channel 17 is in a communication 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 variable volume cavity can suck in liquid through the liquid inlet channel. The pump chamber 16, the liquid inlet channel 17, the liquid outlet channel 18, the liquid inlet control 19 and the liquid outlet control 20 are respectively arranged in two to form two groups of liquid conveying systems. The two liquid outlet channels 18 are communicated with a liquid discharge pipe 21 after converging; the volume change periods of the variable volume chambers of the two groups of liquid delivery systems are opposite. When the volume of the variable-volume cavity of one group of liquid delivery systems is reduced, and the liquid outlet control 20 moves along the liquid outlet cavity channel 18 towards one end far away from the variable-volume cavity; the volume of the variable volume cavity of the other group of liquid delivery system is increased, the liquid outlet control 20 of the other group of liquid delivery system moves along the liquid outlet cavity channel 18 towards one end close to the variable volume cavity, and liquid in the liquid outlet cavity channel 18 flows backwards; at this time, the liquid from the liquid outlet channel 18 of one group of liquid feeding system to the liquid outlet channel 21 compensates the liquid from the liquid outlet channel 18 of the other group of liquid feeding system, and the liquid in the liquid outlet channel 21 is in a stopped state. When the volume of the variable-volume cavity of one group of liquid delivery systems is reduced and the liquid outlet control 20 is kept at one end of the liquid outlet cavity channel 18 far away from the variable-volume cavity; the volume of the variable volume cavity of the other group of liquid delivery system is increased, and the liquid outlet control 20 of the other group of liquid delivery system can be kept at one end of the liquid outlet cavity channel 18 close to the variable volume cavity, so that the liquid outlet cavity channel 18 is kept in a disconnected state, and liquid in the liquid outlet cavity channel 18 stops flowing backwards; at this time, the liquid fed to the liquid discharge pipe 21 from the liquid outlet channel 18 of one of the liquid feeding systems is smoothly discharged through the liquid discharge pipe 21, and the liquid in the liquid discharge pipe 21 is in an outflow state. Therefore, the volume of the variable volume chambers of the two groups of liquid conveying systems can be changed periodically, so that the conveyed oil phase can be switched between the outflow state and the stopped outflow state.
Further, the pump chamber 16 has an opening; the variable capacitance mechanism comprises: a power source 23 for forming a closed diaphragm 22 for the opening and driving the diaphragm 22 to periodically extend towards two sides; wherein the space between the diaphragm 22 and the pump chamber 16 constitutes a variable volume chamber; the pump chamber 16 is fitted with a pump cover 24 at the opening, and the periphery of the diaphragm 22 is sandwiched between the pump chamber 16 and the pump cover 24. The pump cover 24 is provided with a recess on the side facing the membrane 22 to leave room for the membrane 22 to extend in the direction of the pump cover 24. As the diaphragm 22 expands toward the pump chamber 16, the volume of the variable-volume chamber decreases; as the diaphragm 22 expands toward the pump cap 24, the volume of the variable volume 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 comprises: two limiting pieces 25 positioned on two sides of the diaphragm 22, a plug rod 26 fixedly connected with the two limiting pieces 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 portion 29 and an eccentric portion 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 stopper rod 26 so that the stopper rod 26 is limited to move in the longitudinal direction. It will be appreciated that the periodic stretching of the diaphragm 22 towards both sides by the servo motor drive of the above-described transmission mechanism not only allows for precise regulation of the period, but also allows for a higher upper frequency limit for the diaphragm 22 to perform a periodic action.
Further, the liquid inlet control 19 and the liquid outlet control 20 are both spherical. The liquid inlet control 19 and the liquid outlet control 20 are partially or completely made of iron; the liquid inlet channel 17 is provided with a first electromagnet at one part of the outer wall for controlling the initial position of the liquid inlet control 19; the liquid outlet channel 18 is provided with a second electromagnet at one part of the outer wall for controlling the initial position of the liquid outlet control 20. It should be appreciated that the inlet and outlet channels 17, 18 may be made of non-ferrous materials. Before starting the servo motor, electrifying the first electromagnet and the second electromagnet 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 closed, and the servo motor is started, so that the liquid inlet control 19 and the liquid outlet control 20 can be positioned at more accurate positions when the periodical motion is made. Generally, the initial positions of the liquid inlet control 19 and the liquid outlet control 20 of one group of liquid delivery systems are at one end close to the variable-volume cavity, and the initial positions of the liquid inlet control 19 and the liquid outlet control 20 of the other group of liquid delivery systems are at one end far away from the variable-volume cavity.
Further, the inner diameters of the ports at the two ends of the liquid inlet cavity channel 17 are smaller than the diameter of the liquid inlet control 19; the liquid inlet cavity channel 17 comprises a first sealing section 31 and a first communicating section 32 which are in butt joint; the inner diameter of the first sealing section 31 is smaller than or equal to the diameter of the liquid inlet control 19; the first flow-through section 32 has a first main channel 33 adapted to the diameter of the feed control 19, and a first secondary channel 34 for the passage of liquid. It should be appreciated that the inner diameter of the first main channel 33 is the same as the diameter of the feed control 19 or slightly larger than the diameter of the feed control 19. The liquid inlet ports of the liquid inlet channels 17 of the two groups of liquid feeding systems can be communicated with the same liquid inlet pipe. In addition, a first connection portion having a first through hole may be provided at the outer wall of the first closing section 31, a second connection portion having a second through hole may be provided at the outer wall of the first flow passage section 32, a third connection portion having a third through hole may be provided at the outer wall of the inlet pipe end portion, and a corresponding first screw hole may be provided at the outer wall of the pump chamber 16; thus, the liquid inlet pipe, the first closing section 31, the first communicating section 32, and the pump chamber 16 can be connected at one time by inserting 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 sealing section 35 and a second flowing section 36 which are in butt joint; the inner diameter of the second sealing section 35 is smaller than or equal to the diameter of the liquid outlet control 20; the second flow-through section 36 has a second main channel adapted in diameter to the tapping control 20, and a second secondary channel for the passage of liquid. It should be appreciated that the second main channel has an inner diameter that is the same as the diameter of the tapping control 20 or slightly larger than the diameter of the tapping control 20. The structural dimensions of the liquid outlet channel 18 and the liquid inlet channel 17 can be set to be identical; when in installation, the first circulation section 32 of the liquid inlet cavity channel 17 and the second sealing section 35 of the liquid outlet cavity channel 18 are only required to face the variable-volume cavity. In addition, the liquid discharge tube 21 comprises an arc section and a straight section communicated with the middle part 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 is arranged on the outer wall of the second flow section 36, a sixth connecting part with a sixth through hole is arranged on the outer wall of the two ends of the arc section, and a corresponding second threaded hole is arranged on the outer wall of the pump chamber 16; thus, by inserting the bolts, the drain pipe 21, the second closing section 35, the second circulation section 36, and the pump chamber 16 can be connected at one time.
In addition, the inner water phase pump 9 and the outer water phase pump 7 can both adopt the same structural principle as the oil phase pump 4. Of course, other structures can be adopted for the oil phase pump 4, the inner water phase pump 9 and the outer water phase pump 7, so as to realize high-frequency intermittent conveying of the inner water phase, the oil phase and the outer water phase.
While particular embodiments of the present invention have been described above, it will be understood by those skilled in the art that various changes and modifications may be made to these embodiments without departing from the spirit and scope of the 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.
CN202210531865.5A 2022-05-17 2022-05-17 Preparation method of liposome Active CN114887562B (en)

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CN1895223A (en) * 2006-04-05 2007-01-17 沈阳药科大学 Production of elaioplast
CN105727857A (en) * 2014-12-10 2016-07-06 黑龙江鑫达企业集团有限公司 Microfluidic apparatus produced by 3D printing
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