Vesicles and their use for drug delivery

Abstract

To provide a vesicle containing an amphiphilic compound formed by attaching a hydrophilic group to cholesterol.SOLUTION: The vesicle has a hollow shape with an outer shell containing a compound represented by the formula (1) in the figure.SELECTED DRAWING: None

Description

[Technical Field] 【0001】 This invention relates to vesicles containing modified cholesterol and their use for drug delivery. [Background technology] 【0002】 The inventors previously developed a surfactant-like compound characterized by comprising a hydrophilic group, such as ethylene glycol, a hydrophobic group, and 1 to 5 nitrogen-containing groups positioned between them (Patent Document 1). These surfactant-like compounds have low toxicity to living organisms, are small in size, are easily transported to target sites, and can form nucleic acid complexes with high expression efficiency at target sites. It has been reported that when the surfactant-like compound of Patent Document 1 forms a complex with DNA, the gene transfer efficiency of the surfactant-like compound-DNA complex is higher than when DNA alone is administered. [Prior art documents] [Patent Documents] 【0003】 [Patent Document 1] Patent No. 6358661 [Overview of the Initiative] [Problems that the invention aims to solve] 【0004】 It is known that amphiphilic molecules can be self-assembled to form vesicles, which are vesicles with a closed, spherical membrane structure. Vesicles allow for diverse molecular designs and have the potential to exhibit novel functions such as sustained release of substances, capture of toxic substances, and masking of odor and taste molecules. Therefore, their use as carriers in drug delivery systems (DDS), biomaterials, and functional materials is being investigated. In particular, vesicles with a lipid bilayer membrane composed of phospholipids derived from living organisms are called liposomes, and various studies are being conducted on their safety for living organisms. 【0005】 However, Patent Document 1 does not disclose the relationship between the surfactant-like compound and vesicle formation. 【0006】 The problem to be solved by the present invention is to provide a vesicle formed from a compound having a hydrophobic moiety derived from cholesterol and a hydrophilic polymer chain moiety. 【Means for Solving the Problems】 【0007】 The present inventors have found that a hollow vesicle having, as an outer shell, a compound in which a hydrophilic polymer chain having an ether bond is bonded to the 3-position of the sterol skeleton of cholesterol can be formed, and have completed the present invention. 【0008】 Item 1. A vesicle having an outer shell containing the compound represented by formula (1) and having a hollow shape. 【Chemical Formula】 (In the formula, X is 【Chemical Formula】 (In the formula, m is an integer from 2 to 43, and n is from 1 to 3.) Or 【Chemical Formula】 (In the formula, n is from 1 to 3.) is.) 【0009】 Item 2. A drug carrier containing the vesicle according to Item 1. 【0010】 Item 3. A substance encapsulation system containing the vesicle according to Item 1 or 2 and a substance encapsulated in the vesicle. 【0011】 Item 4. The substance encapsulation system according to Item 3, wherein the substance is a drug. 【0012】 Item 5. The compound represented by formula (1) is the compound represented by formula (4) below, and the substance is insulin, the substance encapsulation system according to item 3 for inhibiting amyloid-beta aggregation. [ka] (In the formula, m is an integer between 2 and 43.) 【0013】 Item 6. The compound represented by formula (1) is the compound represented by formula (5) below, and the substance is zinc, the substance-encapsulating system according to item 3 that targets liver cells. [ka] 【0014】 Item 7. Compounds represented by the following formula (5). [ka] [Effects of the Invention] 【0015】 The present invention provides vesicles that are highly safe and have higher structural stability compared to liposomes in which the hydrophobic portion is an alkyl chain, as well as drug carriers and drug delivery systems containing such vesicles. [Brief explanation of the drawing] 【0016】 [Figure 1] Graph showing CMC measurement of Chol-PEG500. [Figure 2] Graph of I1 / I3 versus LogC. [Figure 3] (A) Measurement of average particle size of Chol-PEG500 and Chol-PEG2000 by dynamic light scattering. (B) Schematic diagram of Chol-PEG500. (C) Schematic diagram of Chol-PEG2000. [Figure 4] (A) Transmission electron microscope image of Chol-PEG500. (B) Transmission electron microscope image of Chol-PEG2000. [Figure 5](A) Photograph of the gel after gel electrophoresis of a sample mixed with Chol-PEG500 and insulin. (B) Photograph of the gel after gel electrophoresis of a sample mixed with Chol-PEG2000 and insulin. [Figure 6] (A) Transmission electron microscope image of insulin alone. (B) Transmission electron microscope image of a sample of insulin with Chol-PEG500 added. [Figure 7] (A) Microscopic image of a sample without Chol-PEG500 added. (B) Microscopic image of a sample with 100 equivalents of Chol-PEG500 added. [Figure 8] Graph showing the fluorescence intensity of samples in which Chol-PEG500 was added to aggregated amyloid-beta in various amounts. [Figure 9] (A) Graph of CMC measurement of Chol-Lac. (B) Graph of I1 / I3 vs. LogC. [Figure 10] Particle size of Chol-Lac (solvent: water). [Figure 11] TEM observation image of a Chol-Lac vesicle. [Figure 12] (A) Confocal microscope image of a Chol-Lac vesicle containing Acid Red52, (B) Phase contrast image. [Figure 13] A schematic diagram illustrating the relationship between zinc ion secretion from the pancreas and zinc and insulin release from the liver in healthy individuals (Normal model) and patients with type II diabetes. [Figure 14] (A) Chol-Lac 5.0 × 10⁻² mg / mL (stained with uranium acetate). (B) Zn²⁺ / Chol-Lac 2.5 × 10⁻² mg / mL (stained with uranium acetate). (C) ZnCl₂ (not stained with uranium acetate). [Figure 15] (A) Particle size of Zn2+-encapsulated Chol-Lac in water (4°C), (B) Graph of turbidity of Zn2+-encapsulated Chol-Lac after addition of fetal bovine serum albumin. [Figure 16] A graph showing the cell viability of HepG2 cells after various samples were added. [Figure 17] Evaluation of zinc ions taken up by cells using a fluorescent probe. Zinc ion-encapsulated Chol-Lac was added only to cells (A) and (C), and to cells (B) and (D). Phase contrast is used for (C) and (D). [Figure 18] (A) Graph showing zinc ion uptake into Hep2 cells and C2C12 cells, respectively. (B) Graph showing zinc ion uptake in Hep2 cells when inhibitors are added at various concentrations. [Modes for carrying out the invention] 【0017】 Embodiments of the present invention are described below. 【0018】 In this specification, "vesicle" refers to a substance having an outer shell formed by the self-assembly of amphiphilic molecules, and having a space inside the outer shell, thus having a hollow shape. 【0019】 The vesicle of the embodiment of the present invention is a hollow vesicle having an outer shell containing a compound represented by formula (1). 【0020】 [ka] 【0021】 In the formula, X is 【0022】 [ka] 【0023】 (In the formula, m is an integer between 2 and 43, and n is an integer between 1 and 3.) or 【0024】 [ka] 【0025】 (In the formula, n is between 1 and 3.) That is the case. 【0026】 The compound represented by formula (1) is a compound formed by linking cholesterol and a hydrophilic polymer having an ether bond via an amide bond. The compound represented by formula (1) is amphiphilic because it has both a hydrophilic and a hydrophobic part. When such amphiphilic molecules self-assemble, the hydrophilic part derived from the hydrophilic polymer chain faces outward from the outer shell, while the hydrophobic part derived from cholesterol forms a spherical lipid membrane, and voids are formed inside the spherical lipid membrane. 【0027】 The hydrophilic group represented by formula (2) has repeating units of polyoxyethylene. If the value of n is too small, the compound represented by formula (1) cannot have amphiphilic properties and cannot form vesicles. Also, if the value of m is too large, the compound represented by formula (1) cannot form a lipid bilayer and forms micelles instead of vesicles. For this reason, m is between 2 and 43. Preferably, m is between 11 and 13. n is between 1 and 3, preferably n is 2. When n is 2 in the hydrophilic group represented by formula (2), the following formula (2a) is obtained. 【0028】 [ka] 【0029】 (In the formula, m is an integer between 2 and 43.) The molecular weight of the hydrophilic group represented by (2) is preferably 100 to 1800, and more preferably 200 to 1500. When X of the compound represented by formula (1) is the hydrophilic group represented by formula (2), the molecular weight of the compound represented by formula (1) is preferably 480 to 2200, and more preferably 480 to 1900. 【0030】 The hydrophilic group represented by formula (3) has an alkylene bonded to the 1-position of the glucose unit of lactose via an amino group. n is 1 to 3, preferably n is 2. When n is 2 in the hydrophilic group represented by formula (3), the following formula (3a) is obtained. 【0031】 [ka] 【0032】 The proportion of the compound represented by formula (1) in the amphiphilic molecules within the vesicles is not particularly limited, but from the viewpoint of ease of vesicle formation, it is preferably 50% by mass or more, and may be 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 100% by mass. Furthermore, it is preferably 50 mol% or more in mole fraction, and may be 60 mol% or more, 70 mol% or more, 80 mol% or more, 90 mol% or more, or 100 mol%. 【0033】 The compound represented by formula (1) in the amphiphilic molecule within the vesicle may be one type, or a combination of two or more compounds represented by formula (1). 【0034】 Vesicles may contain other components such as forming aids, stabilizers, and binders, as long as their structure is not broken. 【0035】 Formation aids are not particularly limited and include phospholipids, monoglycerides, diglycerides, polyglycerol fatty acid esters, sterols, polyoxyalkylene sterols, fatty acid esters of sterols, fatty acids, polyoxyalkylene hydrogenated castor oil, and polyoxyalkylene castor oil. 【0036】 Examples of stabilizers include synthetic polymers such as acrylic polymers, vinyl polymers, and polyoxyalkylenes, semi-synthetic polymers such as cellulose derivatives, modified starch, and lignin derivatives, and natural polymers. Stabilizers are mainly added to water and have the effect of improving the storage stability of vesicles and microcapsules in the dispersion medium by increasing viscosity, etc. 【0037】 The particle size of the vesicles in the embodiments of the present invention is not particularly limited, but is, for example, 100 nm or larger. The upper limit is not particularly limited, but is, for example, 1000 nm or less. 【0038】 The particle size of vesicles can be measured individually using a transmission electron microscope. Alternatively, the average particle size of vesicles can be determined from the particle size distribution measured by laser diffraction and dynamic light scattering using a commercially available laser diffraction / dynamic light scattering particle size distribution analyzer. 【0039】 A method for producing a hollow vesicle having an outer shell containing a compound represented by formula (1) of the present invention will be described. 【0040】 In one embodiment, the compound represented by formula (1) can be produced by known methods. For example, the compound represented by formula (1), in which X is a hydrophilic group represented by formula (2), can be obtained by dissolving PEG-NH2 and cholesteryl chloroformate in chloroform, adding triethylamine and heating to react, and then purifying the mixture, as described in S. Asayama et al., Bioconjugate Chem. 2018, 29, 67. 【0041】 Vesicles can be easily formed simply by preparing an aqueous solution of the compound represented by the obtained formula (1) at room temperature. At this time, sonication is applied to encapsulate the substance in the hollow part (internal aqueous phase) of the vesicle. 【0042】 In another embodiment, the compound represented by formula (1), in which X is a hydrophilic group represented by formula (3), is obtained by mixing a DMF solution of lactose with a DMF solution of a compound (EtdNH2-Chol) obtained by reacting cholesteryl chloroformate with alkylenediamine (e.g., ethylenediamine), incubating at room temperature for 1 day, then adding a DMF solution of NaBH3CN to this mixture, incubating at room temperature for 5 days, and then dialyzing. 【0043】 Next, vesicles are prepared in aqueous solution by ultrasonically treating the solution in the dialysis membrane after dialysis using the compound represented by formula (1). The ultrasonic treatment is performed with an output of 50-200W, a frequency of 10-100KHz, a temperature of 4-50℃, and a time of 1 minute to 3 hours, using the above compound, and 5.4 × 10 -3 mg / mL ~ 1.8 × 10 -1 This can be done within a concentration range of mg / mL. 【0044】 The vesicles of the embodiments of the present invention can be used as carriers for encapsulating substances. In this specification, "encapsulation" of a substance includes the incorporation of the substance into the internal aqueous phase of the vesicle, the incorporation of the substance into an outer shell formed by a compound represented by formula (1), and bonding of the substance to the outer shell. 【0045】 The vesicles of the embodiments of the present invention have high structural stability compared to liposomes, where the hydrophobic portion is an alkyl chain, because the hydrophobic portion is derived from cholesterol. Furthermore, the vesicles of the embodiments of the present invention are preferable because they are easy to prepare, as they can be considered as a single film formed of a bilayer of the compound, simply by mixing the compound represented by formula (1) in water and subjecting it to sonication. 【0046】 The vesicles of the embodiments of the present invention can be applied to a wide range of fields, such as drug delivery, cosmetics, pharmaceuticals, fragrances, pesticides, and inks, because they can encapsulate a large amount of substance (e.g., drugs) in their hollow portion (internal aqueous phase). Examples of substances include drugs, fluorescent molecules, peptides, metals (including ions of elemental metals and metal compounds), and proteins. Furthermore, hydrophobic substances can be incorporated into or bound to the hydrophobic portion of the outer shell that partitions the hollow portion, and hydrophilic substances can be incorporated into or bound to the hydrophilic portion of the outer shell. 【0047】 The substance encapsulation system of the embodiment of the present invention includes the vesicle described above and the substance encapsulated within the vesicle. Examples of the substance include drugs, fluorescent molecules, peptides, metals (including ions of elemental metals and metal compounds), and proteins. 【0048】 The substance encapsulation system of the embodiment of the present invention may be a drug delivery system comprising the vesicles described above and a drug encapsulated within the vesicles. 【0049】 In the above drug delivery system, the compound represented by (1) is the compound represented by the following formula (4), and the drug may be insulin. Such a drug delivery system can be used as a drug delivery system for inhibiting amyloid-beta aggregation or for promoting the refolding of aggregated amyloid-beta. 【0050】 [ka] 【0051】 (In the formula, m is an integer between 2 and 43.) In the above drug delivery system, the compound represented by formula (1) is the compound represented by formula (5) below, and the drug may be zinc. Such a drug delivery system can be used as a drug delivery system targeting hepatocytes and is expected to serve as a platform for DDS carriers. 【0052】 [ka] 【0053】 Embodiments of the present invention include compounds represented by the following formula (5). 【0054】 [ka] 【0055】 As described above, the compound represented by formula (5) can be used in the production of vesicles according to embodiments of the present invention. Vesicles produced using such a compound represented by formula (5) have higher structural stability compared to liposomes in which the hydrophobic portion is an alkyl chain. 【0056】 The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Examples] 【0057】 Example 1: Chol-PEG 500 synthesis As described in S. Asayama et al., Bioconjugate Chem. 2018, 29, 67, 66.81 mg (0.01499 mol) of PEG-NH2 (molecular weight of PEG: 500) was dissolved in 10 mL of chloroform, and 4 μL of triethylamine (TEA) and 129.49 mg (0.28884 mmol) of cholesteryl chloroformate were added. The mixture was reacted at 40°C for 24 hours with stirring. Distilled water was added to the reaction product to separate the TEA, and the product was purified by distillation and filtration to obtain cholesteryl-terminated PEG (molecular weight 913, hereafter referred to as Chol-PEG). 500 (As shown) (Yield approximately 50%) was obtained. The synthesis was completed. 1 This was confirmed by 1H-NMR measurement. 【0058】 Similarly, by using PEG-NH2 (PEG molecular weight: 2000) instead of PEG-NH2 (PEG molecular weight: 500) and the above manufacturing method, Chol-PEG 2000 I obtained it. 【0059】 [ka] 【0060】 Example 2: Chol-PEG 500 Measurement of critical micelle concentration (CMC) Chol-PEG was detected by the pyrene-based fluorescent probe method according to the method described in ED Goddard, et al, Langmuir. 1985, 1,3,352-355. 500CMC measurement was performed. The first peak and the third peak of the spectrum in Figure 1 are denoted as I1 and I3, and from the values of I1 / I3 for each plotted concentration, it was shown that the CMC was very low, at 0.11 - 11 mM (Figure 2). 【0061】 Example 3: Measurement of particle size The particle sizes of Chol-PEG 500、 Chol-PEG 2000 at concentrations above their respective CMCs were measured. Chol-PEG 500 showed a very large average particle size of about 500 nm (Figure 3(A), left graph), suggesting that vesicles were formed (Figure 3(B)). On the other hand, Chol-PEG 2000 showed an average particle size of about 10 - 20 nm (Figure 3(A), right graph), suggesting that micelles were formed (Figure 3(C)). 【0062】 Example 4: Transmission electron microscope observation Transmission electron microscopy (TEM) observations were carried out to confirm vesicle formation. Chol-PEG 500、 Chol-PEG 2000 was adjusted to 2.90 mM and diluted 1 / 100 immediately before use. 2 μL of the sample was added onto the grid and left to dry naturally for two nights. Then, 2 μL of the staining agent (2% uranyl acetate) was added and blotted with filter paper. Observation was carried out at an accelerating voltage of 120 kV. In the normal protocol, 2% uranyl acetate is added immediately after placing the sample on the grid, but this time, since vesicle formation was expected, a system of natural drying was used so that the structure would not be damaged during the staining operation. For Chol-PEG 500 particle sizes consistent with the DLS measurement results (Figure 3(A)) were observed, indicating that vesicles were formed (Figure 4(A)). On the other hand, since Chol-PEG 2000 was below the CMC, no particles were observed (Figure 4(B)). 【0063】 Example 5: Insulin encapsulation by vesicles 50 μg of insulin (10 mg / mL insulin in 25 mM HEPES, pH 8.2) and Chol-PEG500 7.96 μg (1 equivalent), 15.92 μg (2 equivalents), 39.80 μg (5 equivalents), 79.60 μg (10 equivalents), 159.2 μg (20 equivalents), 238.80 μg (30 equivalents), 318.40 μg (40 equivalents), and 398.00 μg (50 equivalents) were diluted to a total volume of 125 μL in pH 7.4 50 mM potassium phosphate buffer, and sonication (110 W, 40 kHz) was performed for 5 minutes. An 8% gel was prepared and electrophoresis was performed at 20 mA for 45 minutes. After electrophoresis, the gel was shaken with fixative, then stained with CBB stain for 30 minutes, and the changes in the bands were observed. 【0064】 Chol-PEG 500 In all samples to which Chol-PEG was added, 500 Compared to the Naked insulin sample without Chol-PEG (circled in a square), the band was observed to be shifted towards the higher molecular weight side (Figure 5(A)). 500 In the sample where 30 equivalents of Chol-PEG were added to insulin in a molar ratio of 1 (third lane from the right), a significant band shift towards the high molecular weight side was observed. From this, it was inferred that insulin was encapsulated within the Chol-PEG vesicles. On the other hand, in the same Chol-PEG test performed under the same conditions, 2000 No band shift was observed in either the gel electrophoresis results for insulin (Figure 5(B)). 【0065】 Example 6: Transmission electron microscopy observation of insulin-encapsulated vesicles Chol-PEG 500 To confirm that insulin is encapsulated within the vesicles, a sample identical to that used in gel electrophoresis in Example 5 was diluted to 1 / 100, added to a grid in 2 μL, and allowed to air dry for two nights. Then, 2 μL of staining agent (2% uranyl acetate) was added and absorbed with filter paper. The sample was observed using a transmission electron microscope at an accelerating voltage of 120 kV. As shown in Figure 6(A), the insulin alone does not exhibit a uniform particle size, indicating a flexible structure. On the other hand, as shown in Figure 6(B), Chol-PEG... 500In samples to which 30 equivalents were added, no flexible naked insulin was observed; instead, particles with a diameter of several hundred nanometers were observed. This confirmed that all of the insulin was encapsulated in vesicles. 【0066】 Example 7: Chol-PEG 500 Inhibition of amyloid-beta aggregation by vesicles First, following the protocol of the amyloid-beta protein manufacturer (Peptide Institute), amyloid-beta aggregation was initiated by incubating a potassium phosphate buffer solution of amyloid-beta in a microplate at 37°C for 48 hours. Subsequently, 100 equivalents of Chol-PEG were added to the aggregated amyloid-beta in a molar ratio. 500 After adding the substance and incubating at 37°C for 24 hours, the fluorescent dye thioflavin (ThT) was added, and observation was performed using a fluorescence microscope (Ex: 442 nm, Em: 485 nm). ThT is known to specifically bind to the β-sheets of aggregated amyloid-β, increasing its fluorescence intensity. Chol-PEG 500 Microscopic images of samples without the addition of Chol-PEG showed green dots, confirming the presence of aggregated amyloid-beta (Figure 7(A)). 500 When 100 equivalents of were added, almost no green fluorescence was observed in the microscopic image (Figure 7(B)). 【0067】 To quantify this, we measured the fluorescence intensity (Ex: 442 nm, Em: 485 nm) using a microplate reader, and found that Chol-PEG 500 In the sample to which 100 equivalents of [substance name] were added, the fluorescence intensity was significantly lower compared to amyloid-beta alone (Figure 8), confirming that aggregation had been refolded. 【0068】 Chol-PEG 500 It was successfully demonstrated that this substance has a dual function: not only does it act as a drug carrier by encapsulating insulin, but it also has a refolding effect on aggregated amyloid-beta through interaction with it. 【0069】 Example 8: Synthesis of Chol-Lac A 2 mL DMF solution of lactose (0.253 mmol) and a 2 mL DMF solution of EtdNH2-Chol (0.077 mmol) were mixed and incubated at room temperature for 1 day. Next, a 1 mL DMF solution of NaBH3CN (0.074 mmol) was added to this mixture and incubated at room temperature for 5 days, after which it was dialyzed to obtain Chol-Lac. After dialyzing, vesicles were formed by sonication of the solution within the dialysate membrane. 【0070】 [ka] 【0071】 Example 9 Confirmation of vesicle formation of Chol-Lac Fluorescence measurements were performed using pyrene, similar to Example 2. The fluorescence intensity of pyrene was measured by varying the concentration of the Chol-Lac solution, yielding the spectrum shown in Figure 9(A). The first and third peaks of this spectrum were denoted as I1 and I3, and the intensity ratio of I1 / I3 was plotted on the vertical axis and Log(concentration) on the horizontal axis, resulting in Figure 9(B). The concentration was 5.4 × 10⁻⁶. -3 The intensity ratio decreased when the concentration was above mg / mL. A decrease in the intensity ratio is generally said to indicate that pyrene has been incorporated into the hydrophobic field. Therefore, this experiment suggests that above a certain concentration, Chol-Lac forms aggregates that have a hydrophobic field. 【0072】 Next, DLS measurements were performed. From the pyrene experimental results, the particle size of Chol-Lac vesicles was 150 nm or less at the concentration at which aggregates with hydrophobic fields were formed (Figure 10). This value can be said to be an appropriate size for DDS carriers. In the experiments to be evaluated in the future, the concentration of Chol-Lac vesicles will be set to 5.4 × 10⁻⁶. -2 The concentration was set to mg / mL. 【0073】 Furthermore, TEM measurements were performed. Grids were prepared, dried for two days, and then stained with uranium acetate. The observed images are shown in Figure 11. This figure shows that Chol-Lac is spherical, suggesting that the experiments so far have shown that Chol-Lac forms spherical vesicles or micelles. 【0074】 Finally, to determine whether it was a vesicle or a micelle, we attempted to encapsulate Acid Red 52, a hydrophilic fluorescent reagent, into Chol-Lac by sonication for 1.5 hours. After dialysis of the prepared solution for 4 days, we observed it under a confocal microscope. As a result, fluorescence was observed inside (Figure 12(A), (B)). Therefore, the retention of the hydrophilic reagent inside confirmed that Chol-Lac has an internal aqueous layer and forms a vesicle structure. 【0075】 [ka] 【0076】 Example 10 Zn 2+ Zn for delivery systems 2+ Preparation of cell-targeting vesicles Zinc is one of the physiologically active essential trace elements and has many functions in the body, one of which is the function of suppressing insulin breakdown in the liver. In diabetic patients, compared to healthy individuals, the pancreas does not secrete zinc ions properly, resulting in zinc deficiency in the liver and excessive breakdown of insulin (Figure 13). Therefore, it is thought that being able to directly deliver zinc ions to the liver could be a new treatment for diabetes. 【0077】 Therefore, using the Chol-Lac synthesized in Example 9, zinc ions (Zn 2+ We attempted to develop a carrier for delivering zinc ions to the liver. Our goal was to efficiently deliver zinc ions to the liver by having the β-galactose at the lactose terminus of the Chol-Lac molecule specifically interact with the asialoclycoprotein receptor in the liver. 【0078】 Zn ion encapsulation in Chol-Lac occurs in Chol-Lac solution (concentration 5.4 × 10⁻⁶). -2 After sonicating a zinc chloride aqueous solution (mg / mL) with Chol-Lac for 1.5 hours, unencapsulated zinc ions were removed by dialysis (molecular weight cutoff = 1000). Table 1 shows the results of atomic absorption spectroscopy of the solution within the dialysis membrane after dialysis. The system with Chol-Lac mixed showed higher absorbance than the control with zinc alone. 【0079】 [Table 1] 【0080】 Example 11 Zn 2+ TEM observation of internal vesicles TEM imaging shows that the zinc ions (Zn) obtained by Chol-Lac in Example 10 are visible. 2+ The encapsulation of zinc ions was visually confirmed. 3.0 μL of the sample was added to a hydrophilic-treated grid and allowed to air dry for 2 days. After 2 days, 2.0 μL of uranyl acetate was added, and after 3 seconds, the uranyl acetate was removed with paper and observed by TEM. Chol-Lac containing zinc ions (Figure 14(B)) was stained more internally than Chol-Lac without zinc ions (Figure 14(A)). Furthermore, the particle size was larger than that of the control with only zinc chloride solution (Figure 14(C)), suggesting that Chol-Lac contains zinc ions. 【0081】 Zn 2+ The encapsulated Chol-Lac showed no change in particle size over time in water at 4°C for one month, indicating its stability over time (Figure 15(A)). Also, Zn 2+ The encapsulated Chol-Lac did not show turbidity in serum proteins, indicating that it also possesses serum stability (Figure 15(B)). 【0082】 Example 12: Cell Experiment Since this study targets the liver, cell experiments were performed using HepG2 cells, a human liver model cell. First, cytotoxicity was evaluated using the Alamer Blue assay. Specifically, 1.0 × 10⁶ HepG2 cells were placed in one well. 4 The samples were seeded in microplates at the specified concentrations and incubated at 37°C for 24 hours (in a 5% CO2 incubator). Next, 50 μL of sample (physiological saline, Chol-Lac vesicles, or Zn) was added per well. 2+ The encapsulated Chol-Lac vesicles (solvent: physiological saline) were added and incubated for 24 hours. After washing with physiological saline, 100 μL of Alamar Blue was added and incubated for 2 hours. Subsequently, 80 μL was transferred to a black plate and fluorescence was measured using a microplate reader (Ex. 550 nm, Em. 595 nm). 【0083】 The results are shown in Figure 16. Compared to the control with only physiological saline (100% cell viability), Chol-Lac vesicles and Zn 2+ Both encapsulated Chol-Lac vesicles showed cell viability of over 85%, indicating low cytotoxicity. 【0084】 Example 13 Zn 2+ Enhanced intracellular zinc ion uptake by encapsulated Chol-Lac Next, Zn was taken into the cell. 2+ Ions were evaluated using a fluorescent probe method. ZnAF-2DA was used as the fluorescent reagent. This reagent exhibits green fluorescence upon reaction with zinc ions. Specifically, 1.5 × 10⁶ HepG2 cells were placed on an 8-well glass slide. 5 Seeds were individually seeded and incubated at 37°C for 24 hours (in a 5% CO2 incubator). 75 μL of culture medium and sample (Zn) were placed in each well. 2+75 μL of encapsulated Chol-Lac vesicles were added to each well and incubated at 37°C for 24 hours (in a 5% CO2 incubator). The medium was removed, and after washing twice with physiological saline, 150 μL of 5 μM ZnAF-2DA was added to each well and incubated at 37°C for 2 hours (in a 5% CO2 incubator). The ZnAF-2DA was removed, and after washing twice with physiological saline, 100 μL of DAPI (1 / 10000) was added to each well and incubated in the dark for 30 minutes. The DAPI was removed and washed twice with physiological saline. The dividers on the slide glass were removed, and one drop of 200 μL of 50% glycerol was added to each well using a pipette, and a coverslip was attached. The cells were then observed using a confocal laser scanning microscope. 【0085】 As a result, as shown in Figures 17(B) and (D), cells treated with zinc ion-encapsulated Chol-Lac showed stronger green fluorescence compared to the control cells shown in Figures 17(A) and (C). Therefore, it was confirmed that zinc ions were released from the Chol-Lac vesicles and taken up into the cells. 【0086】 [ka] 【0087】 Example 14: Involvement of asialocsaccharide receptors in the uptake of Zn ions into cells HepG2 cells or C2C12 cells are placed in 5.0 × 10⁶ wells. 5 The sample was seeded at the specified concentration onto a microplate and incubated at 37°C for 24 hours (in a 5% CO2 incubator). The culture medium was removed, and 250 μL of fresh medium and 220 μL of sample (Zn) were added to each well. 2+ or Zn 2+The cells were inoculated with encapsulated Chol-Lac vesicles and incubated at 37°C for 24 hours (in a 5% CO2 incubator). The culture medium was removed, the cells were washed twice with physiological saline, and 450 μL of 2% TritonX-100 solution was added to each well. After incubation overnight, the solution was transferred to a microtube, 900 μL of purified water and 150 μL of 1M HCl were added to prepare 1.5 mL of cell lysate, and the amount of zinc ions taken up into the cells was quantitatively evaluated by atomic absorption spectrometry. 【0088】 The results are shown in Figure 18(A). Zn 2+ Compared to adding Zn as a single element 2+ In the system using encapsulated Chol-Lac, high Zn uptake was obtained in both HepG2 and C2C12 cells. Furthermore, compared to C2C12 cells, which lack hepatic asialoglycoprotein heterogeneity, HepG2 cells showed higher Zn uptake. 2+ The increased uptake suggests that zinc can be efficiently delivered via receptors in the liver. 【0089】 Furthermore, to evaluate that uptake was via receptors, inhibition experiments were performed using asialofetuin reagent. In the above tests, 220 μL of sample (Zn) was used per well. 2+ Instead of adding encapsulated Chol-Lac vesicles and incubating at 37°C for 24 hours (in a 5% CO2 incubator), various concentrations of asialofetin were added to 260 μL of sample and incubated at 4°C for 4 hours. 【0090】 The added asialofetwin binds to asialoclycoprotein receptors on the cell surface, thus allowing Zn to enter the cell via the receptor. 2+ It inhibits the uptake of Zn. 2+ The amount of uptake decreased in a concentration-dependent manner of asialofetwin (Figure 18(B)). Therefore, Zn 2+ The results suggest that encapsulated Chol-Lac is efficiently taken up via asialocsaccharide receptors. 【0091】 Based on the above, the Chol-Lac synthesized in this embodiment forms a vesicle structure and can encapsulate zinc within the vesicle, and Zn 2+ It was suggested that the encapsulated Chol-Lac vesicles are taken up in the liver via asialoglycoprotein receptors. Therefore, Chol-Lac vesicles are considered promising as a zinc ion delivery carrier that targets the liver for the treatment of diabetes.

Claims

[Claim 1] A vesicle having a hollow shape and an outer shell containing a compound represented by the following formula (1). 【Chemistry 1】 (In the formula, X is 【Chemistry 2】 (In the formula, m is an integer between 2 and 13, and n is an integer between 1 and 3.) or 【Transformation 3】 (In the formula, n is between 1 and 3.) (That is the case.) [Claim 2] A drug carrier comprising the vesicle described in claim 1. [Claim 3] A substance encapsulation system comprising a vesicle according to claim 1 or 2 and a substance encapsulated within the vesicle. [Claim 4] The substance encapsulation system according to claim 3, wherein the substance is a drug. [Claim 5] The compound represented by formula (1) is the compound represented by formula (4) below, and the substance is insulin, the substance encapsulation system according to claim 3 for inhibiting amyloid-beta aggregation. 【Chemistry 4】 (In the formula, m is an integer between 2 and 13.) [Claim 6] The substance encapsulation system according to claim 3, wherein the compound represented by formula (1) is the compound represented by the following formula (5), and the substance is zinc, and targets liver cells. 【Transformation 5】 [Claim 7] The compound represented by the following formula (5). 【Transformation 6】