KR20230155923A - Bicelle and preparation method thereof - Google Patents

Abstract

The present invention provides a bicell that combines two types of lipids in a specific ratio and has excellent material preservation and delivery properties compared to conventional liposomes or micelles due to its disk-shaped morphological characteristics, and a method of manufacturing this bicell. Regarding,
It relates to a bicell comprising a first lipid forming a plate-like bilayer structure and a second lipid bonded to the side of the bilayer to prevent exposure of the hydrophobic tail portion formed in the first lipid.

Description

BICELLE and its manufacturing method {BICELLE AND PREPARATION METHOD THEREOF}

The present invention provides a bicell that combines two types of lipids in a specific ratio and has excellent material preservation and delivery properties compared to conventional liposomes or micelles due to its disk-shaped morphological characteristics, and a method of manufacturing this bicell. It's about.

Representative lipid carriers include micelles and liposomes.

When a surfactant is placed in an aqueous solution, a micelle is a spherical aggregate formed by the hydrophilic group being exposed to the outside to form a surface in contact with water and the hydrophobic group forming a nucleus inward, forming a colloidal dispersed state in water. do. With this structure, water-insoluble ingredients are contained and dissolved in the center of micelles where fat-soluble substances are clustered together, and the hydrophilic molecules of the amphiphilic molecules surrounding them are located on the outside, thereby maintaining the water-insoluble molecules dissolved in water. can do.

Liposomes are spherical vesicle structures made of one or more artificially created lipid bilayers, and are used to deliver nutrients or drugs, and can be used to encapsulate unstable substances.

However, micelles have high permeability through barriers due to their small size, but have the disadvantage of low encapsulation efficiency and low protective effect for contained substances.

In addition, liposomes have a relatively high protective effect for substances incorporated into the double layer, but have problems such as poor physical stability, low penetration efficiency, and loss through blood circulation.

Republic of Korea Patent Publication No. 10-2021-0106607 (Publication date: 2021.08.31)

The problem to be solved by the present invention is to provide a bicell capable of inserting various bioactive substances such as cell membrane proteins, peptides, DNA, and RNA, and a method of manufacturing the bicell.

Another object of the present invention is to provide a vicelle and a method of manufacturing the bicell that overcome the problems of liposome-, vesicle-, or micelle-type delivery systems.

Another object of the present invention is to provide a bicell with high utility as a transdermal and mucosal delivery vehicle and a method for manufacturing the bicell.

The problems to be solved through various embodiments of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In the present invention, two types of lipids are combined in a specific ratio, so that one lipid forms a double layer and is assembled into a disk shape, and the other type of lipid can be stabilized by binding to the side.

In particular, stability can be further improved by including additional additives.

In addition, rapid mixing between solvent and buffer can be induced using microfluidic devices, microfluidizers, dispermixes, homomixers, etc.

Specific details of other embodiments are included in the detailed description and drawings.

According to embodiments of the present invention, the bicell produced has excellent stability,

In particular, when using microfluidic devices, it may be possible to set the ratio of the two lipids that make up the bicell within a wider range. In addition, due to the pre-assembly of the double layer, the film has excellent physical properties and can be manufactured under mild conditions.

According to embodiments of the present invention, the uniformity and stability of the manufactured bicell can be improved, and continuous production is possible.

Additionally, due to the above characteristics, various research and commercial applications such as pharmaceuticals, cosmetics, and functional foods may be possible.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.

1 is a TEM image of a bicell according to the present invention.
Figure 2 is a schematic diagram of one embodiment of a method for manufacturing a bicell according to the present invention.
Figure 3 is a graph showing the particle size of the material prepared through electron microscope analysis according to the present invention.
Figure 4 is a graph showing the film properties of the material prepared through electron microscope analysis according to the present invention.
Figure 5 is a detailed view of a microfluidic device according to the present invention.
Figure 6 is a process diagram of another embodiment of the bicell manufacturing method according to the present invention.
Figure 7 is a schematic diagram showing the synthesis process of a bicell using a microfluidic device according to the present invention.
Figure 8 is a graph showing the particle size of bicell according to the present invention.
Figure 9 is a graph comparing the particle properties of the bicell according to the present invention.
Figure 10 is a graph comparing the mucosal layer permeability of Bicell according to the present invention.

Hereinafter, embodiments will be described in detail with reference to the attached drawings. However, since various changes can be made to the embodiments, the scope of rights of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes for the embodiments are included in the scope of rights.

The terms used in the examples are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless clearly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

In addition, when describing with reference to the accompanying drawings, identical components will be assigned the same reference numerals regardless of the drawing symbols, and overlapping descriptions thereof will be omitted. When describing an embodiment, if it is determined that a detailed description of related known technology may unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

Additionally, when describing components of the embodiment, terms such as first, second, A, B, (a), and (b) may be used. These terms are only used to distinguish the component from other components, and the nature, order, or order of the component is not limited by the term. When a component is described as being “connected,” “coupled,” or “connected” to another component, that component may be directly connected or connected to that other component, but there is no additional component between each component. It should be understood that may be “connected,” “combined,” or “connected.”

Components included in one embodiment and components including common functions will be described using the same names in other embodiments. Unless stated to the contrary, the description given in one embodiment may be applied to other embodiments, and detailed description will be omitted to the extent of overlap.

The present invention relates to a bicelle and a method of manufacturing the same, wherein the bicelle is bonded to a first lipid forming a plate-like bilayer structure and a side of the bilayer to prevent exposure of the hydrophobic portion formed in the first lipid. It is a structure containing secondary lipids.

Both the first lipid and the second lipid are composed of a hydrophilic head portion and a hydrophobic tail portion, and the hydrophobic tail portions of the first lipid may be bonded to each other vertically to form a plate-shaped double layer structure. In other words, only the hydrophilic head portion of the first lipid is exposed at the top and bottom.

At this time, the second lipid is bound to the side of the bilayer so that the hydrophobic tail portion of the first lipid is not exposed. More precisely, the hydrophobic tail portion of the second lipid is combined laterally with respect to the bilayer so that it is located inside the bilayer, and as a result, the outer surface of the produced bicell is entirely composed of a hydrophilic head portion, and the hydrophobic tail portion is located on the inside. It forms a cake-shaped (disk-shaped) structure.

Therefore, the functional material can be stored, protected and transported in the space formed inside the bicell, and then discharged at a designated location so that the function of the functional material can be expressed.

In addition, the length of the hydrophobic tail portion of the first lipid is formed to be longer than the length of the hydrophobic tail portion of the second lipid, so that binding stability can be increased when the second lipid is bound to the side of the bilayer.

In addition, these bicells have a rigid double-layer structure, but can also have a small size of 5 to 50 nm in diameter and a disk-shaped morphological feature.

In one embodiment of the present invention, the first lipid may include at least one of saturated fatty acids or unsaturated fatty acids having 14 to 18 carbon atoms.

More preferably, it may contain a saturated fatty acid with 14 to 16 carbon atoms or an unsaturated fatty acid with 18 carbon atoms, and may further include lecithin.

Lecithin is one of the phospholipids containing glycerin phosphate and is soluble in alcohol and ether. It can also be hydrolyzed to produce choline, phosphoric acid, glycerol, and fatty acids, and the fatty acids can mainly be stearic acid, palmitic acid, oleic acid, and linoleic acid.

In the lecithin, choline can form a hydrophilic head portion and fatty acid can form a hydrophobic tail portion.

The saturated fatty acid may include one or more selected from the group consisting of Myristic acid, Palmitic acid, and Stearic acid.

The unsaturated fatty acid may include one or more selected from the group having single and multiple double bonds, such as palmitoleic acid and myristoleic acid.

The second lipid may include a surfactant or a phospholipid, and the surfactant may include a single chain structure, a double chain structure, or a cholesteryl structure.

That is, the second lipid may include any one or more of a surfactant containing a single chain structure, a surfactant containing a double chain structure, and a surfactant containing a cholesteryl structure.

The surfactant containing the single chain structure may include one or more selected from the group consisting of Cetrimonium bromide (CTAB), Sucrose Stearate, and Polysorbate. .

The surfactant containing the double chain structure includes at least one selected from the group consisting of Dihexanoyl Phosphatidylcholine, Diheptanoyl Phosphatidylcholine, Dioctanoyl Phosphatidylcholine, and Phosphatidylglycerol. can do.

The surfactant containing the cholesteryl structure may include one or more of cholesteryl ether and CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate). .

Additionally, additional additives may be added to improve the stability of the manufactured bicell, and the additive may be one of cholesterol and ceramide or a mixture of the two.

When the above additives are further combined, the effects of controlling drug release ability, stabilizing membranes, and improving the physical properties of biological barriers can be expected.

Furthermore, [Table 1] below is a table showing the success of bicell production according to the type of the first lipid (domain lipid) and the second lipid (surfactant).

[Table 1]

The present invention can provide a method for manufacturing the bicell as described above.

As an example of the method for manufacturing the bicell, as shown in Figure 2,

1. Inject chloroform into the flask and add DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) 10~14mM, DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) 6~ Dissolving 10mM;

2. Dissolving 1 to 10% by weight of the total phospholipid concentration with a fat-soluble substance containing TECA {Centella asiatica quantitative extract} or curcumin;

3. Producing a phospholipid thin film in the flask under the conditions of a water bath temperature of 40-80°C and 110-130 rpm;

4. Degassing for 8 to 10 hours to remove solvent;

5. Hydrating the produced phospholipid thin film by injecting a pH 7.0, 10mM concentration of PBS (Phosphate Buffered Saline) buffer into the flask;

6. 40~60 , heating and stirring under conditions of 230 to 260 rpm;

의 냉동고에서 냉각하는 단계;7. -70~90 below zero

Cooling in a freezer;

8. Preparing a Bicell solution by repeating steps 6 and 7 above;

냉장 환경에서 8~10시간 안정화하는 단계;9. 3~5 times the prepared bicell solution

Stabilizing in a refrigerated environment for 8 to 10 hours;

It can be done with

Another example of the method for manufacturing the bicell is to manufacture a microfluidic device, as shown in FIGS. 5 to 7,

1. Preparing an inner phase solution by dissolving saturated fatty acids or unsaturated fatty acids consisting of 12 to 18 carbon chains of 20 to 100 mM or more, or both, using alcohol including methanol, ethanol, and isopropyl alcohol;

2. Dissolving 1 to 10% by weight of the total phospholipid concentration with a fat-soluble substance containing TECA {Centella asiatica quantitative extract} or curcumin;

3. Preparing an outer phase solution by dissolving 5 to 40mM of phospholipid or surfactant with 5 to 8 carbon atoms in pH 7.0, PBS buffer;

4. Injecting the inner phase solution and the outer phase solution into the microfluidic device through different injection ports;

5. A step in which bicelles are synthesized by diffusion of solvent and buffer in the cross-flow path within the microfluidic device;

It can be done with

At this time, in the case of bicell manufacturing conditions using microfluidic devices, the total flow rate (TFR) was set to 4,000~100,000㎕/h and the flow rate ratio (FRR) was set to 10~20, and the inner flow rate (FRR) was set accordingly. The flow rates of the phase and outer phase can be 100~1000㎕/h and 4000~8,000㎕/h, respectively.

In particular, when using microfluidic devices, the ratio of the two lipids that make up the bicell can be carried out in a wider range, the membrane physical properties are excellent due to the pre-assembly of the double layer, and manufacturing can be possible even under mild conditions.

Based on the above example, a bicell was prepared as follows.

Manufacturing example 1.

1. Inject chloroform into a round bottom flask and dissolve 12mM of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and 8mM of DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine). TECA was dissolved together at 1/10 of the phospholipid concentration.

2. After attaching the flask to the evaporator, a phospholipid thin film was produced under the conditions of a water bath temperature of 60°C and 120 rpm.

3. The solvent was completely removed through an additional degassing process of more than 8 hours.

4. The prepared phospholipid thin film was hydrated by injecting a pH 7.0, 10mM PBS buffer solution into the flask.

5. Heating and stirring was performed under the conditions of 50°C and 250 rpm using a constant temperature water bath, and cooled in a -80°C freezer.

6. The heating, stirring and cooling processes were performed sequentially for 15 minutes and repeated 5 times.

7. The prepared Bicell solution was stabilized in a refrigerated environment at 4°C for more than 8 hours.

In the bicell solution prepared by the above production method, the formation of bicelles was determined through particle size, membrane properties, and electron microscope analysis.

Unlike liposomes, bicells have a diameter of several nanometers to tens of nanometers. Accordingly, it can be seen that the size shown in Figure 3 corresponds to a bicell or micelle.

Figure 4 shows the membrane physical properties, where the horizontal axis represents the fluidity of the membrane and the vertical axis represents the polarity of the membrane. When the membrane fluidity value is 4 or less and the membrane polarity value is 0.3 or more, it can be viewed as a gel.

In the case of micelles, the membrane properties are significantly lower than those of bicells, so it can be confirmed through Figures 3 and 4 that the manufactured particles are bicells.

Manufacturing example 2.

1. An inner phase solution was prepared by dissolving 100mM of saturated fatty acid consisting of 16 carbon chains using methanol, and TECA was dissolved at 1/10 of the phospholipid concentration.

2. An outer phase solution was prepared by dissolving 25mM of 7-carbon phospholipid and surfactant in pH 7.0, PBS buffer.

3. The solutions were injected into the microfluidic device to synthesize a bicell.

Manufacturing Example 2 is to manufacture a bicell according to the process shown in FIG. 6 using the microfluidic device shown in FIG. 5. As shown in FIG. 7, the inner phase solution and the outer phase solution are introduced into the microfluidic device. Then, it spreads as shown in Figure 7 to produce a bicell.

Figure 8 shows the particle size of the formed bicell, and it can be seen that the concentration becomes thicker around 10 nm, which is smaller than the particle produced by the batch process in Preparation Example 1. The size difference from Preparation Example 1 can be attributed to the improved homogeneity of the double layer membrane and the increase in the number of lipids forming the edge as the assembly sequence was controlled using a microfluidic device. The improvement in the homogeneity of the double-layer membrane leads to the improvement of the membrane's physical properties, which means that, as shown in Figure 9, compared to the bi-cell manufactured by the batch process, the bi-cell manufactured through a microfluidic device has higher membrane polarity (Fig. 9b) and lower membrane fluidity (Fig. 9c). ) showed the results.

Figure 10 shows the permeability of Bicell to the artificial mucosal layer. In particular, it compares liposomes and Bicell, and it was confirmed that Bicell showed high permeability to the artificial mucosal layer.

Additionally, the microfluidic device of Preparation Example 2 can be manufactured as follows (see Figure 6).

Manufacturing example 3. Manufacturing of microfluidic devices

1. A photolithography process is used to produce a master mold using a silicon wafer, and the channel is replicated by soft-molding using PDMS (Polydimethylsiloxane) and bonded to a plate made of the same material to produce microfluidic fluid. Produce devices

2. The microfluidic device for bicell manufacturing has a uniform size of 100 ~ 200㎛ in height and width, and uses a silicon wafer-based master mold through a photolithography process to mix PDMS (Polydimethylsiloxane), a thermosetting polymer, with a curing agent and 10 µm. : After mixing at a 1 ratio, it was cured by going through a degassing process and heating at 70°C for 1 hour.

3. The cured PDMS was released from the mold and bonded to a plate made of PDMS to produce a microfluidic device.

4. The fabricated microfluidic device includes an injection part capable of injecting phospholipids of various lengths and a mixing part capable of mixing the injected samples, and the injection part may be composed of two channels depending on the type of sample.

Additionally, a bicell was manufactured as in Example 1 below and its aspects were analyzed.

Example 1.

For the production of Vicelle, methanol, one of the alcohols used as the inner phase, was mixed with 10 mM DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine, 14:0 PC), a long-chain phospholipid, respectively. 20mM, 100mM, 150mM, and 200mM were dissolved and the bicell formation pattern according to concentration was analyzed.

1. In the outer phase, which is a buffer solution, dissolve 20mM of DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine, 6:0 PC) so that it naturally binds to the hydrophobic group of the DMPC bilayer (DMPC fragment) formed in the mixing section. induced.

2. It was confirmed that the alcohol solution and buffer solution as solvents meet in the cross passage of the microfluidic device to form a laminar flow, and that rapid and uniform mixing is achieved by mutual diffusion between the two phases.

3. DMPC, which was dispersed in an alcohol environment, a non-polar solvent, was exposed to a polar environment by mixing alcohol and buffer solution and naturally formed phospholipid self-assembly, ultimately forming a small-sized phospholipid bilayer.

4. DHPC dispersed in the buffer defected to the hydrophobic group of the DMPC double-layer self-assembly to form a rim, forming a stable disk-shaped bicell.

5. Bicell shape analysis was conducted according to changes in DMPC concentration.

The total flow rate is 6,000㎕/h, the alcohol sample containing DMPC is set at 300㎕/h, the buffer sample containing DHPC is set at 5,700㎕/h, and the conditions are set to FRR (Flow Rate Ratio) = 19. did.

As a result of the analysis, it was confirmed that spherical liposomes were formed in samples with a DMPC concentration of 50mM or less, and that disc-shaped bicelles with a diameter of less than 15nm were formed in samples with a DMPC concentration of 50mM or more.

In the case of the Bicell solution, when observed with the naked eye, it is produced as a transparent solution with a slightly milky color. As a result of shape analysis through transmission electron microscopy (TEM) imaging (see Figure 1), a disk-shaped phospholipid complex was produced, and the size was It was found to have a horizontal length of about 15 to 25 nm.

The phospholipid mixing ratio that can be manufactured by Bicell can be q - value = 0.1 ~ 2, and the final concentration of phospholipids can be 15 ~ 30mM. It was found that the preferred diameter range of bicells that can be manufactured under the above conditions is 10 to 50 nm.

As described above, although the embodiments have been described with reference to limited drawings, those skilled in the art can apply various technical modifications and variations based on the above.

For example, the described techniques are performed in a different order than the described method, and/or components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or other components are used. Alternatively, appropriate results may be achieved even if substituted or substituted by an equivalent.

Therefore, other implementations, other embodiments, and things equivalent to the claims should also fall within the scope of the claims described below.

B: Bicell

Claims (9)

The first lipid forming a plate-shaped double layer structure;
a second lipid bound to the side of the bilayer to prevent exposure of the hydrophobic tail formed in the first lipid;
Bicell containing. According to claim 1,
A bicell in which the length of the hydrophobic tail portion formed in the first lipid is longer than the length of the hydrophobic tail portion formed in the second lipid. According to claim 1,
The first lipid is a bicell containing at least one of saturated fatty acids or unsaturated fatty acids having 14 to 18 carbon atoms. According to claim 1,
The second lipid is a bicell containing at least one of a surfactant containing a single chain structure, a surfactant containing a double chain structure, and a surfactant containing a cholesteryl structure. According to claim 1,
The bicell is a bicell consisting of a cake-shaped structure with a diameter of 5 to 50 nm. According to claim 4,
The surfactant containing the single chain structure is a bicell containing at least one selected from the group consisting of Cetrimonium bromide (CTAB), Sucrose Stearate, and Polysorbate. . According to claim 4,
The surfactant containing the double chain structure includes at least one selected from the group consisting of Dihexanoyl Phosphatidylcholine, Diheptanoyl Phosphatidylcholine, Dioctanoyl Phosphatidylcholine, and Phosphatidylglycerol. Bycell. According to claim 4,
The surfactant containing the cholesteryl structure includes at least one of cholesteryl ether and CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate). Preparing an inner phase solution to form a first lipid by dissolving saturated fatty acids, unsaturated fatty acids, or both using alcohol;
Dissolving 1 to 10% by weight of the total phospholipid concentration with a fat-soluble substance containing TECA {Centella asiatica quantitative extract} or curcumin;
Preparing an outer phase solution to form a second lipid by dissolving phospholipids or surfactants in a pH 7.0, PBS buffer solution;
Injecting the inner phase solution and the outer phase solution into the microfluidic device;
synthesizing a bicell by diffusion of the buffer solution in a cross-flow path within a microfluidic device;
A method for producing a bicell comprising: