Complex lipid-containing complex and composition containing said complex

Abstract

Provided are: lipid-containing nanoparticles that replace a reconstituted high-density lipoprotein (rHDL); and a composition comprising said lipid-containing nanoparticles. This complex comprises a complex lipid and an apolipoprotein A-I-derived helix 4 (H4) peptide, wherein the H4 peptide has an amino acid sequence of YLDDFQKKWQEEMELYRQKVE (SEQ NO. 1) and has a C-terminus that may be amidated. This composition comprises a complex according to the present invention. The composition can be for cosmetic use or percutaneous absorption drug use, or for the treatment of inflammatory diseases.

Description

Complex containing complex lipids and composition containing the complex

[0001] The present invention relates to a complex lipid-containing complex and a composition comprising the complex.

[0002] Lipoproteins, which are micellar complexes composed of lipid-binding proteins collectively known as apolipoproteins and lipids, are present in human blood and function as lipid transporters in the blood. High-density lipoproteins (HDL) can be artificially reconstituted using apolipoprotein AI (apoA-I) and glycerophospholipids. Reconstituted high-density lipoproteins (rHDL) are nanoparticles with a diameter of less than approximately 100 nm, and are being developed for use as pharmaceuticals, for example (Patent Documents 1 and 2).

[0003] ApoA-I has a structure consisting of ten linked amphipathic α-helices, and the hydrophobic face of each helix binds to lipids to form rHDL nanoparticles. It is known that peptides containing the sequences of the first and tenth α-helices of apoA-I react with glycerophospholipids to form HDL-like particles (nanodiscs, NDs) (Non-Patent Document 1). However, it was not known that peptides in the α-helix regions other than the first and tenth helices possessed ND-forming properties.

[0004] WO2016 / 104690 JP 2021-138629 A

[0005] Leman, LJ et al., J. Med. Chem. 2014, 57, 2169-2196.

[0006] The present invention aims to provide lipid-containing nanoparticles that can replace rHDL and compositions containing the lipid-containing nanoparticles.

[0007] In order to achieve the above object, the present inventors searched for peptides capable of forming NDs by a method other than that described in Non-Patent Document 1. As a result, they found that the fourth helix (H4) peptide of the ten α-helices that constitute apoA-I has the ability to form NDs, and further found that NDs (H4-NDs) can be formed from the H4 peptide and a complex lipid containing a glycerophospholipid, thereby completing the present invention.

[0008] The present invention is as follows. [1] A complex comprising a complex lipid and apolipoprotein AI-derived helix 4 (H4) peptide, wherein the H4 peptide has the amino acid sequence YLDDFQKKWQEEMELYRQKVE (SEQ NO. 1) and is optionally amidated at the C-terminus. [2] The complex according to [1], wherein the molar ratio of the complex lipid to the H4 peptide (complex lipid:peptide H4) is in the range of 5 to 500:1. [3] The complex according to [1] or [2], wherein the complex lipid is a glycerolipid or a sphingolipid. [4] The complex according to [3], wherein the glycerolipid is a glycerophospholipid or a glyceroglycolipid. [5] The complex according to any one of [1] to [4], wherein the complex has an average particle size in the range of 8 to 100 nm. [6] The complex according to any one of [1] to [5], wherein the complex is a disc-shaped particle. [7] A composition containing the complex according to any one of [1] to [6]. [8] The composition according to [7], which is for use in cosmetics or transdermal medicine. [9] The composition according to [8], which further contains a component usable as a cosmetic or transdermal medicine.

[10] The composition according to [9], which is for use in treating inflammatory diseases.

[11] The composition according to

[10] , which is for use in treating systemic inflammatory diseases.

[12] The composition according to

[10] or

[11] , which further contains an anti-inflammatory component.

[0009] [A] Use of the conjugate according to any one of [1] to [5] for the production of cosmetics. [B] Use of the conjugate according to any one of [1] to [5] for the production of a transdermal drug. [C] Use of the conjugate according to any one of [1] to [5] for the production of a therapeutic drug for inflammatory diseases. [D] Use of the conjugate according to any one of [1] to [5] for the production of a therapeutic drug for systemic inflammatory diseases. [E] The conjugate according to any one of [1] to [5] for use in cosmetics. [F] The conjugate according to any one of [1] to [5] for use in a transdermal drug. [G] The conjugate according to any one of [1] to [5] for use in a therapeutic drug for inflammatory diseases. [H] The conjugate according to any one of [1] to [5] for use in the treatment of systemic inflammatory diseases. [I] Use of the conjugate according to any one of [1] to [5] for the treatment of inflammatory diseases. [J] Use of the conjugate according to any one of [1] to [5] for the treatment of systemic inflammatory diseases. [K] A method for treating an inflammatory disease, comprising administering the conjugate according to any one of [1] to [5] to a subject. [L] A method for treating a systemic inflammatory disease, comprising administering the complex according to any one of [1] to [5] to a subject.

[0010] According to the present invention, it is possible to provide a novel lipid-containing nanoparticle that can replace rHDL. Furthermore, according to the present invention, it is possible to provide a nanoparticle complex that has anti-inflammatory activity equal to or greater than that of rHDL and has superior cellular absorbability.

[0011] (a) Size distribution of H4-ND and (b) atomic force microscope image. (a) Change in the average particle size of H4-ND over time (0.5, 1, 2, 4, 8, 24 h) after standing at each temperature is shown. The horizontal axis represents the elapsed time (h), and the vertical axis represents the volume average particle size (nm). (b) Photographs of samples 8 and 24 h after standing at 30 and 37°C are shown (clear precipitation is evident from 8 h onwards). (c) Change in peptide concentration over time in the supernatant of the sample solution when stood at 4 and 37°C is shown. Change in the peak top wavelength of tryptophan (Trp) fluorescence in H4-ND over time, as measured using a fluorometer, is shown. The horizontal axis represents the incubation time (h), and the vertical axis represents the peak top wavelength of Trp fluorescence (nm). (a) The uptake of each lipid nanoparticle containing fluorescent lipid (Rhod-PE) into human umbilical vein endothelial cells (HUVEC) was examined using a confocal fluorescence microscope. Confocal microscopy images taken 1 h after the start of treatment with each lipid nanoparticle are shown. The scale bar indicates 20 μm. (b) The intracellular Rhod-PE weight was quantified and normalized to intracellular protein. Each bar indicates the mean, and the error bars indicate the standard deviation. H4-ND is shown in red, and rHDL is shown in black. (n=4) *p < 0.05 by Tukey-Kramer test (H4-ND vs. rHDL). (a) The uptake of each lipid nanoparticle into human monocyte (THP-1)-derived macrophages was examined using a confocal fluorescence microscope. Confocal microscopy images taken 1 h after the start of treatment with each lipid nanoparticle are shown. The scale bar indicates 20 μm. (b) The intracellular Rhod-PE weight was quantified and normalized to intracellular protein. Each bar indicates the mean, and the error bars indicate the standard deviation. H4-ND is shown in red, and rHDL is shown in black. (n=4) *p < 0.05 by Tukey-Kramer test (H4-ND vs. rHDL). (a) Fluorescence-activated cell sorting (FACS) analysis of the inhibitory activity of H4-ND on tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule 1 (VCAM-1) expression in HUVECs. Cells were treated with 300 μg peptide or protein / mL. (b) Quantitative analysis of (a) is shown.Data are presented as mean ± standard deviation (N = 4). ELISA analysis results for anti-inflammatory activity against lipopolysaccharide (LPS)-induced TNF-α secretion are shown. Data are presented as mean ± standard deviation (N = 4). In the bar graph, red indicates H4-ND and black indicates rHDL. Filled bars indicate results with LPS treatment, while striped bars indicate results without LPS treatment. The horizontal axis indicates the peptide or protein concentration (μg / mL) in the treated lipid nanoparticles, and the vertical axis indicates the secreted TNF-α concentration (pg / mL). (a) Each lipid nanoparticle was administered via the tail vein of a mouse. Blood was collected at the specified time points and centrifuged to separate plasma and blood cell components. Rhod-PE in the plasma was quantified, and the percentage of the administered dose (%ID / mL) was calculated. Each point represents the mean, and error bars represent standard deviation. The horizontal axis represents time (h), and the vertical axis represents the ratio of the weight of Rhod-PE in the collected blood to the weight of administered Rhod-PE (%ID / mL). (b) The calculated ratio of the amount of Rhod-PE contained in plasma components of blood to the amount of Rhod-PE bound to blood cell components is shown. In both graphs, red indicates H4-ND and black indicates rHDL. In (b), the striped bar indicates the blood cell fraction and the filled bar indicates the plasma fraction. The amino acid sequence of human apoA-I (243 aa) is shown (SEQ ID NO: 2). Each α-helix is ​​underlined (H2 and H3 are contiguous, so H2 is double underlined to distinguish them). The N-terminal side is H1, and the C-terminal side is H10. The fourth bold part is H4.

[0012] The present invention relates to a complex comprising a complex lipid and an H4 peptide derived from apolipoprotein AI, wherein the H4 peptide has the amino acid sequence YLDDFQKKWQEEMELYRQKVE (SEQ NO. 1) and may be amidated at the C-terminus.

[0013] As shown in the amino acid sequence of Figure 9, apolipoprotein AI has a structure in which 10 amphipathic α-helices are linked together, and the hydrophobic face of each helix is ​​thought to bind to lipids. The H4 peptide derived from apolipoprotein AI is the fourth helix peptide, consists of 21 amino acids, and has the amino acid sequence YLDDFQKKWQEEMELYRQKVE (SEQ NO. 1). The C-terminus of the H4 peptide may be amidated.

[0014] The H4 peptide may have one or two amino acids substituted to the extent that ND-forming ability can be maintained. However, from the viewpoint of maintaining ND-forming ability, it is preferable that the amino acids before and after the substitution are amino acids with the same chemical properties, and for example, substitution between amino acids having aromatic side chains (phenylalanine, tryptophan, tyrosine), substitution between amino acids having aliphatic side chains (alanine, isoleucine, leucine, methionine, valine), substitution between amino acids having polar uncharged side chains (asparagine, cysteine, glutamine, serine, threonine), or substitution between amino acids having charged side chains (acidic (aspartic acid, glutamic acid), basic (arginine, histidine, lysine)).

[0015] The H4 peptide may have one or two amino acids deleted or added, as long as the ND-forming ability is maintained. The deletion may be one or two amino acids at either end. The H4 peptide may also have one, two, or three amino acids added at either end, as long as the ND-forming ability is maintained. If the added amino acid is an amino acid with a high affinity for lipids, for example, an amino acid with an aromatic side chain (phenylalanine, tryptophan, tyrosine) or an amino acid with an aliphatic side chain (alanine, isoleucine, leucine, methionine, valine), this may be preferable for maintaining the ND-forming ability.

[0016] The H4 peptide is amidated at its C-terminus to form -CONH ２ The H4 peptide with an amidated C-terminus can maintain its ND-forming ability.

[0017] The "complex lipid" as a component refers to a glycerolipid or a sphingolipid. Glycerolipids include glycerophospholipids and glyceroglycolipids that have a glycerin backbone. Glycerophospholipids refer to glycerolipids that have one or more phosphate ester moieties, and glyceroglycolipids refer to glycerolipids that have a sugar at one of the two phosphate ester moieties. Sphingolipids include sphingophospholipids and sphingoglycolipids that have a sphingosine backbone.

[0018] The glycerophospholipids of the present invention include glycerophospholipids generally known to be contained in natural lipoproteins. Glycerophospholipids known to be contained in high-density lipoproteins (HDL) are preferred, but are not limited thereto. The glycerophospholipid is preferably a long-chain glycerophospholipid containing an acyl group having about 12 to about 18 carbon atoms, preferably about 14 to about 18 carbon atoms. Examples include phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine, with phosphatidylcholine being preferred. Typical examples include, but are not limited to, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), egg phosphatidylcholine (PC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC).

[0019] Glycolipids are lipids to which sugars are bound, and include glyceroglycolipids in which long-chain fatty acids and sugars are bound via glycerol, galactolipids in which the sugar is galactose, and sphingoglycolipids in which long-chain fatty acids and sugars are bound via sphingosine, etc. Examples of glycolipids include digalactosyldiacylglycerol, galactocerebroside, and ganglioside.

[0020] The complex lipids can be used alone or in combination of two or more.

[0021] By using the complex lipid of the present invention, for example, the hardness of the lipid membrane can be adjusted, and further, a stabilized complex can be formed. In addition, by using the complex lipid, the complex can encapsulate a compound such as a drug.

[0022] Cholesterol can also be added to the complex lipid. By incorporating cholesterol into the complex lipid, the hardness of the lipid membrane formed by the complex lipid can be adjusted. By adding cholesterol to the complex lipid, it is possible to interact with active ingredients such as drugs and adjust the encapsulation efficiency into the lipid membrane. The amount of cholesterol added to the complex lipid is preferably in the range of 0.01 to 30 mol%, for example.

[0023] Alternatively, a charged lipid can be added to the complex lipid. By adding a charged lipid, the gene can be bound to H4-ND. The amount of charged lipid added to the complex lipid is preferably in the range of, for example, 0.01 to 30 mol%.

[0024] In the complex of the present invention, the molar ratio of complex lipid to H4 peptide can be, for example, in the range of 5 to 500: 1. From the viewpoint of forming stable nanoparticles, the molar ratio of complex lipid to H4 peptide can be, for example, in the range of 6 to 100: 1, 7 to 50: 1, 8 to 40: 1, 9 to 30: 1, or 10 to 20: 1. However, the molar ratio can be appropriately adjusted depending on the type of complex lipid.

[0025] The composite of the present invention may have an average particle size in the range of 8 to 100 nm. The average particle size may be, for example, in the range of 10 to 70 nm, 10 to 50 nm, 12 to 40 nm, or 14 to 30 nm. Furthermore, the composite of the present invention may be in the form of disc-shaped particles, and the disc-shaped particles may be disc-shaped particles having an average particle size in the above range.

[0026] <Method for producing the complex> The complex of the present invention can be prepared, for example, by a method using sodium cholate. Specifically, the complex lipid micelle dispersion obtained by mixing sodium cholate and complex lipid is mixed with H4 peptide, the resulting mixture is left standing for a certain period of time, and then dialyzed through a dialysis membrane to remove sodium cholate. The reaction solution inside the dialysis membrane is centrifuged, and the lipid nanoparticles contained in the supernatant are purified by density gradient ultracentrifugation to obtain the complex of the present invention. For the method using sodium cholate, for example, see the description in Matz, CE and Jonas, A., J. Biol. Chem. 1982, 257, 4535-4540.

[0027] The complex lipid dispersion can be a PBS solution, and the H4 peptide dispersion can also be a PBS solution. Urea (e.g., 1-6 M) can be added to the H4 peptide PBS solution to improve solubility and / or induce α-helix formation during dialysis. In density gradient ultracentrifugation, the complex of the present invention can be obtained, for example, from the density fraction of 1.063 g / mL to 1.21 g / mL.

[0028] <Composition> The present invention includes a composition containing the complex of the present invention. The composition of the present invention may be a cosmetic composition, a transdermal pharmaceutical composition, or a composition for treating an inflammatory disease.

[0029] The cosmetic composition of the present invention may further contain one or more cosmetic ingredients that can be used as cosmetics. There are no particular restrictions on the cosmetic ingredients.

[0030] The transdermal medicinal composition of the present invention may further contain one or more transdermal medicinal ingredients that can be used as a transdermal drug. There are no particular limitations on the transdermal medicinal ingredients.

[0031] The composition for treating inflammatory diseases of the present invention, which contains the complex of the present invention, exhibits a therapeutic effect for inflammatory diseases due to the inclusion of the complex of the present invention. The composition for treating inflammatory diseases of the present invention can be a composition for treating systemic inflammatory diseases. Furthermore, the composition for treating inflammatory diseases of the present invention can further contain one or more anti-inflammatory ingredients. There are no particular limitations on the anti-inflammatory ingredients, but examples include cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone, loxoprofen sodium, aspirin, celecoxib, diclofenac sodium, naproxen, etc.

[0032] The composition of the present invention can contain, in addition to the complex of the present invention, cosmetic ingredients and transdermal medicinal ingredients, for example, oily ingredients, active ingredients, viscosity modifiers, colorants, fragrances, etc. The composition for the treatment of inflammatory diseases of the present invention can also contain, as additive ingredients, oily ingredients, active ingredients, viscosity modifiers, colorants, fragrances, etc.

[0033] Examples of oily components include hydrocarbons such as squalane and liquid paraffin, vegetable oils such as olive oil, macadamia nut oil, and jojoba oil, animal oils such as beef tallow, esters such as glyceryl triisooctanoate, isopropyl myristate, cetyl isooctanoate, and isooctyl palmitate, silicones such as dimethyl silicone, phenylmethyl silicone, and cyclomethicone, and ultraviolet absorbers such as ethylhexyl methoxycinnamate, octocrylene, ethylhexyl salicylate, homosalate, and polysilicone-15.

[0034] Examples of active ingredients include whitening agents such as ascorbic acid, magnesium ascorbyl phosphate, ascorbyl palmitate, ascorbyl stearate, ascorbyl tetraisopalmitate, ascorbic acid glucoside, arbutin, ellagic acid, and rucinol; NMF components such as amino acids; skin roughness prevention agents such as water-soluble collagen, elastin, glycyrrhizic acid, glycyrrhetinic acid, and ceramide; anti-aging agents such as retinol, vitamin A acid, and astaxanthin; and various vitamins and their derivatives.

[0035] Examples of moisturizing ingredients include polyethylene glycol, propylene glycol, 1,3-butylene glycol, dipropylene glycol, glycerin, sorbitol, xylitol, maltitol, chondroitin sulfate, hyaluronic acid, and sodium pyrrolidone carboxylate.

[0036] The composition of the present invention can contain components that can generally be incorporated into liposomes, as long as the effects of the product of the present invention are not impaired. Examples include higher alcohols, higher fatty acids, and polar lipids. Examples of higher alcohols include lauryl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, and cetostearyl alcohol. Examples of higher fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, and isostearic acid. Examples of polar lipids include sphingolipids, ceramides, cholesterol, and its derivatives.

[0037] The composition of the present invention can be in the form of, for example, a lotion, emulsion, gel, cream, hair treatment, ointment, injection, or the like.

[0038] The present invention will be described in more detail below with reference to examples. However, the examples are merely illustrative of the present invention and are not intended to limit the present invention.

[0039] Example 1-1 Preparation of H4 Peptide H4 peptide was synthesized using a fully automated microwave peptide synthesizer, Biotage® Initiator+ Alstra™ Peptide Synthesizer (Biotage Japan Ltd., Tokyo, Japan). The synthesized peptide was purified using a COSMOIL 5C18-AR-II (20 mm ID, 150 mm) column, yielding H4 peptide with a purity of over 90%. Mass spectrometry detected a peak at m / z = 935.4573, which coincided with the molecular weight of the C-terminally amidated H4 peptide, 2806.15 (M+3H)3+.

[0040] Example 1-2: Preparation of Lipid Nanoparticles and Fluorescently Labeled Lipid Nanoparticles Lipid nanoparticles (H4-ND) composed of H4 peptide and DSPC were prepared. First, a 30 mg / mL solution of sodium cholate in PBS was added to DSPC powder (70.6 μL / mg DSPC), and the DSPC was dispersed by bath sonication at 65°C for 10 min. Next, H4 peptide was dissolved in 4 M urea in PBS to a concentration of 1.5 mg / mL and mixed with the DSPC dispersion (DSPC:H4 = 10:1 (mol)). The mixture was incubated at 55°C for 30 min, and then dialyzed overnight at room temperature against 3 L of PBS using a 50 kDa molecular weight cut off (MWCO) dialysis membrane (REPLIGEN Corp., Waltham, US). The reaction solution inside the dialysis membrane was centrifuged (20,000 × g, 24°C, 30 min), and the lipid nanoparticles contained in the supernatant were purified by density gradient ultracentrifugation.

[0041] Density gradient ultracentrifugation was performed as follows: The required amount of solid KBr was weighed into a vial, and the lipid nanoparticle dispersion was added (0.5 g KBr / mL dispersion). The KBr was thoroughly dissolved by vortexing or sonication. This sample dispersion and a KBr / 0.9% NaCl gradient solution (from bottom to top: 1.21 g / mL, 3.0 mL; 1.063 g / mL, 3.8 mL; 1.019 g / mL, 3.3 mL; 1.006 g / mL, 1.2 mL) were layered in a polyamide sealed tube (Eppendorf Himac Technologies Co., Ltd., Ibaraki, Japan). Ultracentrifugation (249,300 × g, 16°C, 2 h) was performed in a Himac CP80NX (Eppendorf Himac Technologies Co., Ltd.) ultracentrifuge using a P70AT rotor (Eppendorf Himac Technologies Co., Ltd., Ibaraki, Japan). After ultracentrifugation, the solution was collected from the 1.063–1.21 g / mL HDL density fraction and concentrated at 5000 × g, 24°C, 15 min using Amicon® Ultra Centrifugal Filters 50 kDa (Merck KGaA). The concentrate was then placed in a 50 kDa MWCO dialysis membrane tube and dialyzed overnight against 3 L of PBS at room temperature. The reaction mixture inside the dialysis membrane was transferred to a microtube and centrifuged at 20,000 × g, 4°C, 15 min. The supernatant was used for subsequent experiments. Except where noted, all subsequent steps were performed at 4°C.

[0042] For the preparation of fluorescently labeled lipid nanoparticles, DSPC and Rhod-PE dissolved in ethanol were mixed at a molar ratio of 100:1, and the ethanol was evaporated under reduced pressure at 55°C using a rotary evaporator R-300 (Nihon BUCHI, Tokyo, Japan) to form a DSPC lipid film, which was then reacted with proteins and peptides. The subsequent procedures were the same as those described above.

[0043] Reference Example 1: Preparation of Lipid Nanoparticles and Fluorescently Labeled Lipid Nanoparticles The rHDL used in this Reference Example was prepared from apoA-I(44-243), a 43-amino acid deletion form of human apoA-I, and DSPC as a comparison with H4-ND. It has been reported that rHDL prepared from apoA-I(44-243) contains helix 10, which is important for HDL bioactivity, just like rHDL containing full-length apoA-I, and that the structural differences between the two types of rHDL are minimal (Rogers, DP et al., Biochemistry 1997, 36, 288-300). As in Example 1-2, a DSPC dispersion was prepared by adding 30 mg / mL sodium cholate in PBS to DSPC powder (70.6 μL / mg DSPC) and mixing it with a 4 M urea solution of apoA-I(44-243) in PBS (1.5 mg / mL) to obtain lipid nanoparticles containing apoA-I(44-243) (DSPC:apoA-I(44-243) = 80:1 (mol:mol)). Fluorescently labeled lipid nanoparticles were also prepared in the same manner as in Example 1-2.

[0044] Test Example 1: Characterization of H4-ND and rHDL The results of the characterization of each lipid nanoparticle prepared in Examples 1-2 and Reference Example 1 are summarized in Table 1. The glycerophospholipid concentration, peptide / protein concentration, and Rhod-PE concentration of each lipid nanoparticle were measured, and the component ratios of the lipid nanoparticles (lipid:protein, mol:mol) were calculated using these values. These values ​​were found to be 19.1±13.4 (H4-ND) and 80.8±6.6 (rHDL), respectively. The ratios (mol%) of Rhod-PE to glycerophospholipid were 1.8±0.40 (H4-ND) and 1.4±0.03 (rHDL). The volume average particle diameter and zeta potential were also measured. The volume average particle diameters were 19.6±3.1 nm (H4-ND) and 12.3±2.3 nm (rHDL). The particle size distribution (a) and atomic force microscope image (b) of H4-ND are shown in Figure 1.

[0045] (a) shows the particle size distribution obtained by dynamic light scattering analysis. H4-ND is shown in red, and rHDL in black. The fluorescently labeled versions of each lipid nanoparticle are shown with dashed lines. The MV of H4-ND (red, solid line) was 19.6 ± 3.1 nm, that of rHDL (black, solid line) was 12.3 ± 2.3 nm, that of fluorescently labeled H4-ND (red, dashed line) was 20.4 ± 5.7 nm, and that of fluorescently labeled rHDL (black, dashed line) was 14.2 ± 2.6 nm. The vertical axis shows frequency (%), and the horizontal axis shows particle diameter (nm). (b) shows an atomic force microscope image (left) and its cross-sectional shape (right). H4-ND, like rHDL, was disc-shaped. The zeta potential was -16.0 ± 4.0 mV (H4-ND) and -7.7 ± 0.11 mV (rHDL). Furthermore, due to the complexation of Rhod-PE, which contains an anionic sulfonic acid group, the zeta potential of the fluorescently labeled product showed a more negative value than that of the unlabeled product.

[0046]

[0047] Test Example 2: Thermal Stability of H4-ND To examine the thermal stability of H4-ND, H4-ND was allowed to stand at 4, 15, 24, 30, and 37°C, and changes in size and peptide concentration in the supernatant were examined. Samples were collected at 0.5, 1, 2, 4, 8, and 24 hours, and size changes were examined by DLS. The results are shown in Figure 2(a). No size changes were observed up to 24 hours at 4 and 15°C, but the size rapidly increased after 4 hours at 24°C and immediately after standing at 30 and 37°C. Furthermore, significant precipitation was observed after 8 hours at 30 and 37°C (Figure 2(b)).

[0048] After DLS measurement, the sample was centrifuged, and the peptide concentration in the supernatant was examined by reverse-phase high-performance liquid chromatography. The results are shown in Figure 2(c). Since the peptide concentration was constant at all temperatures, the precipitate is thought to be an aggregate of lipids and Rhod-PE. This suggests that H4-ND may rapidly disintegrate at high temperatures, resulting in the separation of the H4 peptide and lipid.

[0049] Next, to gain insight into the environment surrounding the H4 peptide during incubation at 37°C, we examined the time course of the fluorescence peak top wavelength of tryptophan (Trp) contained in the H4 peptide. The Trp excitation wavelength was 280 nm. The results are shown in Figure 3. The fluorescence peak top wavelength of Trp residues in proteins reflects the hydrophobicity of the area surrounding Trp, with a red shift indicating hydrophilicity (Mishra VK, Palgunachari MN, Lund-Katz S, Phillips MC, Segrest JP, Anantharamaiah GM. Effect of the arrangement of tandem repeating units of class A amphipathic α-helixes on lipid interactions. Journal of Biological Chemistry. 1995;270(4):1602-1611. doi:10.1074 / jbc.270.4.1602). Incubation at 4°C or 37°C showed a red shift in the fluorescence wavelength over 5 hours in the 37°C incubation. However, no red shift in the fluorescence wavelength was observed in the 4°C incubation. This suggests that the environment surrounding Trp in H4-ND changed from a hydrophobic to a hydrophilic environment upon incubation at 37°C. This result supports the results shown in Figure 2(c).

[0050] Test Example 3: Affinity of H4-ND for Human Vascular Endothelial Cells and Human Monocyte Cells The uptake of lipid nanoparticles labeled with a fluorescent lipid (Rhod-PE) was examined in human vascular endothelial cells (HUVEC) and human monocytic cell (THP-1)-derived macrophages. The results of confocal fluorescence microscopy observation 1 hour after sample treatment (Figures 4(a) and 5(a)) show that the Rhod-PE fluorescence colocalized with the lysosomal fluorescence. This demonstrated that H4-ND was taken up by the cells. Furthermore, an uptake quantification test was performed 1 and 3 hours after the addition of each lipid nanoparticle (rHDL, H4-ND) (Figures 4(b) and 5(b)). Significant uptake of H4-ND was observed compared to rHDL in both cells. THP-1-derived macrophages also took up more H4-ND than rHDL. Furthermore, a significant increase in uptake was observed only for H4-ND from 1 to 3 hours after the addition.

[0051] Test Example 4 Anti-inflammatory Effect of H4-ND The anti-inflammatory activity of H4-ND was examined using HUVECs in which inflammation was induced with the inflammatory cytokine TNF-α. H4-ND exhibited the same level of inhibitory effect as rHDL on the expression of the adhesion factor VCAM-1 protein, whose expression is induced by TNF-α stimulation (Figure 6).

[0052] Next, anti-inflammatory activity was evaluated using LPS-treated THP-1 macrophage cells. TNF-α secretion was used as an index. As shown in Figure 7, H4-ND dose-dependently inhibited TNF-α secretion up to a peptide concentration of 150 μg / mL, and the effect was significantly higher than that of rHDL at 150 μg / mL. This indicates that H4-ND exhibits anti-inflammatory activity equivalent to or greater than that of rHDL in LPS-treated THP-1 macrophages.

[0053] Test Example 5: Blood Retention of H4-ND Each lipid nanoparticle was administered to BALB / cCrSLc mice, and blood was collected at 0.16, 1, 4, 7, and 24 hours. After separation into plasma and blood cell components, the Rhod-PE concentration in each blood component was quantified using RP-HPLC, and the ratio of the weight of Rhod-PE contained in the collected blood to the weight of Rhod-PE administered was calculated (Figure 8(a)). The half-life was calculated from a decay curve created based on the time change in the amount of Rhod-PE remaining in the blood, and the results were rHDL (3.2 h) and H4-ND (1.1 h). H4-ND showed a shorter blood retention than rHDL. The results of Test Example 2 show that H4-ND breaks down at high temperatures, releasing the peptide.

[0054] To investigate the interaction of each lipid nanoparticle with blood cell components, the weight ratio of Rhod-PE in the plasma / blood cell fraction was calculated (Figure 8(b)). As a result, Rhod-PE was detected in the blood cell fraction for all lipid nanoparticles, suggesting binding to blood cell components. However, there was little difference in the abundance ratio between N4-ND and rHDL in both fractions, suggesting that the stronger cell affinity of H4-ND compared to rHDL observed in HUVEC and THP-1 macrophages in vitro was not observed for blood cells in vivo.

[0055] The present invention is useful in fields that utilize complex lipid-containing complexes.

[0056] SEQ NO. 1: Amino acid sequence of H4 peptide SEQ NO. 2: Amino acid sequence of apoA-I

Claims

1. A complex comprising a complex lipid and apolipoprotein AI-derived helix 4 (H4) peptide, wherein the H4 peptide has the amino acid sequence YLDDFQKKWQEEMELYRQKVE (SEQ NO. 1), and the C-terminus may be amidated.

2. The complex according to claim 1, wherein the molar ratio of the complex lipid to the H4 peptide (complex lipid:peptide H4) is in the range of 5 to 500:

1.

3. The complex according to claim 1, wherein the complex lipid is a glycerolipid or a sphingolipid.

4. The complex according to claim 3, wherein the glycerolipid is a glycerophospholipid or a glyceroglycolipid.

5. The composite of claim 1, wherein the composite has an average particle size in the range of 8 to 100 nm.

6. The complex of claim 5, wherein the complex is a disc-shaped particle.

7. A composition containing the complex according to any one of claims 1 to 6.

8. The composition according to claim 7, which is for use in cosmetics or transdermal medicine.

9. The composition according to claim 8, further comprising an ingredient usable as a cosmetic or transdermal drug.

10. The composition according to claim 9, for treating inflammatory diseases.

11. The composition according to claim 10, for treating systemic inflammatory diseases.

12. The composition of claim 10 further comprising an anti-inflammatory ingredient.

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