JP2021020866A - Surface modifier for lipid membrane structure - Google Patents

Abstract

To provide a surface modifier for a lipid membrane structure that is not a PEG lipid, and a lipid membrane structure containing the modifier.SOLUTION: The present invention relates to a PMPC lipid obtained by binding a polymer comprising 2-methacryloyloxyethyl phosphorylcholine (MPC), and a lipid membrane structure comprising the PMPC lipid as a constituent lipid.SELECTED DRAWING: None

Description

The present invention relates to a material for modifying a lipid membrane structure and a lipid membrane structure modified with the material.

Liposomes, which are a type of lipid membrane structure, can be complexed with drugs and have the function of regulating pharmacokinetics such as reducing the toxicity of drugs due to drug administration, and are useful for human administration. It is one of the high carriers. However, when the liposome was administered in blood, it was captured by phagocytic cells in the reticuloendothelial system (RES) such as the liver and spleen, and it was difficult to retain it in the blood for a long time.

In order to avoid the uptake of liposomes by RES and to retain them in the blood for a long time, there is a method of introducing Poly (ethylene glycol) (PEG) -converted lipids (PEG lipids) into the liposomes to three-dimensionally stabilize the liposome surface. It was proposed (Non-Patent Document 1). The presence of hydrophilic polymers (such as PEG) on the surface of the liposomes forms a film of water on the surface, reducing the adsorption of opsonins to the liposomes and reducing the degree of capture by the mononuclear phagocyte system (MPS). Decrease (Non-Patent Document 2). If the liposomes can stay in the blood for a long time, the drug can be targeted to the tumor or the inflamed site. Currently, some long-term blood-retaining liposome preparations, such as Doxil®, are commercially available. As described above, the PEGylated liposome can be expected to have a preferable long-term retention in blood even when administered in vivo.

However, in recent years, it has been found that antibodies using PEG lipid as an antigen are produced, and as a result, it is clear that PEGylated liposomes are excreted from the blood immediately after administration in two or more repeated administrations. (Non-Patent Document 3). This phenomenon is called the ABC (accelerated blood clearance) effect and has become a major problem for the development of PEGylated liposome preparations for repeated administration.

In view of its usefulness as a carrier for PEGylated liposomes, the ABC effect is being improved, while the development of liposome surface modifiers other than PEG lipids is also underway (Non-Patent Document 4). However, at present, almost no modifiers other than PEG lipids are used, and the development of highly usable liposome surface modifiers is awaited.

Klibanov et al., FEBS Lett. 268: 235-237 1990 Senior et al., Biochim. Biophys. Acta 1062: 77-82 1991 Ishida et al., J. Controlled Release 105: 305-317 2005 Allen and Cullis, Adv. Drug. Deliv. Rev. 65: 36-48 2013

In view of the above circumstances, it is an object of the present invention to provide a surface modifier of a lipid membrane structure other than PEG lipid and a lipid membrane structure containing the modifier.

The inventors have developed poly (2-methacryloyloxyethyl liposomecholine): PMPC (Ishihara et al., J Biomater Sci Polym Ed 28: 884-899 2017), which is considered to be highly biocompatible. When a liposome containing the bound lipid as a constituent lipid of the lipid bilayer of the liposome was prepared, it showed high dispersion stability until several months later, and it was confirmed that the liposome modified with PMPC was difficult to aggregate.
The present invention has been completed based on the above findings.

That is, a PMPC lipid obtained by binding a polymer composed of (1) 2-methacryloyloxyethyl phosphorylcholine (MPC) according to the following (1) to (6) of the present invention.
(2) The PMPC lipid according to (1) above, which is obtained by binding poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) of the following formula (1).
(3) A lipid membrane structure containing the PMPC lipid according to (1) or (2) above as a constituent lipid.
(4) The lipid membrane structure according to (3) above, which is a liposome.
(5) The lipid membrane structure according to (3) above, which is a biological membrane.
(6) The lipid membrane structure modifier containing the PMPC lipid according to (1) or (2) above.

According to the present invention, a lipid membrane structure having high biocompatibility and dispersion stability can be produced. Therefore, it is possible to significantly prolong the blood retention time of the liposome preparation containing the PMPC lipid according to the present invention.

Further, since a desired functional group can be introduced into the PMPC lipid according to the present invention, a membrane structure modified with a desired functional group can be produced by using the PMPC as a lipid component. (That is, it has a use as a modifier on the surface of a lipid structure).

Furthermore, since the PMPC lipid according to the present invention can modify the lipid structure in the living body, for example, by introducing a PMPC lipid in which a desired functional group is bound to a biological membrane such as a cell membrane, the biological membrane is introduced. (Ie, it has a use as a modifier for biological membranes).

Analysis results of liposomes modified with 1,2-dipalmitoyl-sn-glycerol (DPG) -PMPC (10, 20, 50, 100). The results of measuring the particle size (Liposome size), PDI (polydispersity index) and zeta potential (zeta potential) of the liposome immediately after preparation are shown. As a control, the same measurement was performed for unmodified liposomes and PEG-modified liposomes. Analysis results of liposomes modified with DPG-PMPC (10, 20, 50, 100). The results of measuring the particle size (Liposome size), PDI (polydispersity index) and zeta potential (zeta potential) of liposomes one month after preparation are shown. As a control, the same measurement was performed for unmodified liposomes and PEG-modified liposomes. Analysis results of liposomes modified with DPG-PMPC (10, 20, 50, 100). The results of measuring the liposome size (Liposome size), PDI (polydispersity index) and zeta potential (zeta potential) 3 months after the preparation are shown. As a control, the same measurement was performed for unmodified liposomes and PEG-modified liposomes. Analysis results of liposomes modified with 1,2-distearoyl-sn-glycerol (DSG) -PMPC (10, 20, 50, 100). The results of measuring the particle size (Liposome size), PDI (polydispersity index) and zeta potential (zeta potential) of the liposome immediately after preparation are shown. As a control, the same measurement was performed for unmodified liposomes. Analysis results of liposomes modified with DSG-PMPC (10, 20, 50, 100). The results of measuring the particle size (Liposome size), PDI (polydispersity index) and zeta potential (zeta potential) of liposomes one month after preparation are shown. As a control, the same measurement was performed for unmodified liposomes. Analysis results of liposomes modified with DSG-PMPC (10, 20, 50, 100). The results of measuring the liposome size (Liposome size), PDI (polydispersity index) and zeta potential (zeta potential) 3 months after the preparation are shown. As a control, the same measurement was performed for unmodified liposomes. Preparation of fluorescently labeled liposomes containing the PMPC lipid of the present invention and adsorption experiments on substrates. Adsorption of liposomes containing PMPC lipids (DSG-PMPC: PMPC10, PMPC20, PMPC50, PMPC100), unmodified liposomes, and PEG-modified liposomes to substrates (planets having a self-assembled monolayer having an amino group formed on the surface). The result of the test is shown. Observation results of cells modified with PMPC lipid fluorescently labeled with fluorescein of the present invention by a confocal laser scanning microscope.

The first embodiment of the present invention is PMPC (poly (2-methacryloyloxyethyl)) (poly (2-methacryloyloxyethyl)) formed by binding a polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC). phosphorylcholine): PMPC)) lipid (hereinafter, also referred to as “PMPC lipid of the present invention”).
More specifically, the PMPC lipid of the present invention is a lipid having a structure (see formula (2)) to which PMPC represented by the following formula (1) is bound.
In formulas (1) and (2), X is a functional group and Y is a lipid moiety. n is an integer of 10 or more and 300 or less, preferably an integer of 10 or more and 200 or less, and more preferably an integer of 10 or more and 150 or less.

The lipid portion of the PMPC lipid of the present invention may be any lipid, including simple lipids, complex lipids and inducible lipids. More specifically, examples of the type of lipid include acylglycerol, phospholipid, glycolipid, sterol, long chain fatty acid, long chain fatty alcohol, glycerin fatty acid ester and the like.

Examples of the acylglycerol include dioleoylglycerol, dilauroylglycerol, dimyristoylationglycerol, dipalmitoylglycerol and distearoylglycerol.
Examples of phospholipids include phosphatidylcholine such as dioleoil phosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, dioleoil phosphatidylglycerol, dilauroylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, and dipalmitoylphosphatidylglycerol. Phosphatidylglycerols such as glycerol and distearoylphosphatidylglycerol, phosphatidylglycerols such as dioleoil phosphatidylethanolamine, dilauroylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoil glycero Phosphoethanolamine, phosphatidylinositol and the like can be mentioned.
Examples of glycolipids include glycolipids such as sphingomyelin, diglycosyl glyceride and digalactosyl diglyceride, and glycosphingolipids such as galactosyl cerebroside and ganglioside.

Examples of sterols include cholesterol, dihydrocholesterol, lanosterol, stigmasterol and sitosterol.
Examples of the long-chain fatty acid or long-chain fatty alcohol include fatty acids having 10 to 20 carbon atoms or their alcohols, and specific examples thereof include palmitic acid, stearic acid, lauric acid, myristic acid, and pendadesilic acid. Examples include fatty acids such as palmitoleic acid, fatty alcohols such as oleyl alcohol, stearyl alcohol, lauric alcohol, and myristic alcohol.

The PMPC lipid of the present invention can be synthesized by polymerizing 2-methacryloyloxyethyl phosphorylcholine (MPC) as a monomer on the lipid moiety. Here, a small amount of any monomer component may be contained with respect to MPC. The polymerization reaction can be carried out by any method that can be selected from the addition polymerization reactions. For example, as a polymerization method, it can be carried out by an atom transfer radical polymerization (ATRP) method, which is one of the living radical polymerization methods. The initiators used here include initiators for continuous activator regeneration (ICAR), activators generated by electron transfer (AGET), and electron transfer. Activators ReGenerated by Electron Transfer (ARGET) is applicable. Further, not limited to the glycerol skeleton, a compound having a saturated or unsaturated alkyl chain can be used.
When the polymerization reaction is carried out by the ATRP method, for example, α-bromo-isobutyl group, chloromethyl group, N-chlorosulfoneamide group and the like are used as starting groups, and as metal catalysts, for example, copper bromide, copper chloride and iodine. Metallic copper such as copper oxide is preferably a ligand containing nitrogen as the ligand, and in particular, one having a large ATRP equilibrium constant, for example, TPMA (Tris (2-pyridylmethyl) amine), Me ₆ TREN (Tris). [2- (dimethylamino) ethyl] amine), Me ₄ cyclam (1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane), etc. can be used.
Hereinafter, the PMPC lipid of the following formula (3) in which the lipid portion is acylglycerol will be described.

Here, synthesis by the atom transfer radical polymerization method will be described. In order to prepare a polymerization initiator, α-bromo-isobutyl bromide is added to diacylglycerose, the reaction is carried out in dicyclomethane / TEA, the reaction product is added to hexane, the precipitate is recovered, and the mixture is washed with water. , Purified and vacuum dried to obtain an initiator (Scheme 1).

The initiator and 2-methacryloyloxyethyl phosphorylcholine are added to methanol / dioxane containing CuBr2, L (+) ascorbic acid and tris (2-pyridylmethyl) amine, and the temperature is 40 ° C. for about 24 hours under an argon atmosphere. , Carry out a polymerization reaction (Scheme 2). Reaction temperatures are available in the range 15 ° C to 45 ° C. The reaction time can be within 24 hours. In addition to CuBr2, metallic copper such as copper iodide can be used. In addition to tris (2-pyridylmethyl) amines, nitrogen-containing ligands can also be used, such as tris [2- (dimethylamino) ethyl] amines and 1,4,8,11-tetramethyl-1,4, 8,11-tetraazacyclotetradecane etc. can be used.

After the polymerization reaction, the reactant is precipitated with ether, the obtained precipitate is dissolved in pure water, dialyzed and then freeze-dried to obtain the desired PMPC lipid (Scheme 3).

The PMPC lipid of the present invention can introduce a desired functional group into "X" of the above formula (3). For example, by introducing a bromine, chlorine, azide group, amino group, carboxyl group, hydroxyl group, thiol group, or reactive substituent at the position of "X", for example, a fluorescent labeling compound (for example, a fluorescent protein, etc.) ), Peptides, proteins, nucleic acids, polysaccharides, etc. can be easily introduced (Ishihara et al., React. Funct. Polymers 119 125-133 2017).

A second embodiment of the present invention is a lipid membrane structure containing the PMPC lipid of the present invention as a constituent lipid (hereinafter, also referred to as “lipid membrane structure of the present invention”).
Here, the "lipid membrane" is a membrane containing lipid as a main component, and is basically a lipid bilayer or a lipid bilayer formed by associating hydrophobic portions with each other and forming two layers. The structure may consist of multiple layers that are laminated several times, or may consist of a single lipid layer that forms a hydrophobic portion inward or outward.
Further, the lipid membrane structure of the present invention may be any, and is not particularly limited, and may be, for example, an artificially prepared lipid membrane structure such as a liposome, which is a naturally occurring lipid membrane structure. It may be a body, for example, a biological membrane (for example, an intracellular small organ such as a cell membrane (primary plasma membrane), a lysosomal membrane, a follicular membrane, a mitochondrial inner / outer membrane, or a Gordi body membrane).
As described above, the PMPC lipid of the present invention can modify the surface of the lipid membrane structure by being incorporated into the lipid membrane structure. Therefore, the agent containing the PMPC lipid of the present invention has a use as a modifier of the lipid membrane structure.

The lipid structure of the present invention can be produced by a well-known method using the desired lipid and the PMPC lipid of the present invention. Such a method is not particularly limited, and examples thereof include a hydration method, an ultrasonic treatment method, an ethanol injection method, an ether injection method, a reverse phase evaporation method, a surfactant method, and a freezing / thawing method. it can.

If this specification is translated into English and contains the singular words "a", "an", and "the", not only the singular, unless the context clearly indicates otherwise. It shall include more than one.
Hereinafter, the present invention will be described with reference to examples, but the present examples are merely examples of embodiments of the present invention, and do not limit the scope of the present invention.

1. 1. Synthesis of Atom Transfer Radical Polymerization Initiator with Long Chain Alkyl Group 1,2-dimyristoyl-sn-glycerol (DMG), 1,2-dipalmitoyl-sn-glycerol (DPG) and 1,2 according to Scheme 1 above. By reacting -distearoyl-sn-glycerol (DSG) with 2-bromoisobutyryl bromide (BIBB), three types of atom transfer radical polymerization (ATRP) initiators having long-chain alkyl groups with different chain lengths (DMG-Br respectively) , DPG-Br and DSG-Br) were synthesized.
Place the stirrer chip in an eggplant flask, weigh 500 mg of each long chain alkyl glycerol (DMG: 0.98 mmol, DPG: 0.88 mmol, DSG: 0.80 mmol), dissolve in 4.0 mL of dichloromethane, and dissolve 1.1 equivalents of triethylamine ( TEA, 101.19) (DMG synthesis: 110 mg, DPG: 98.0 mg, DSG: 89.0 mg) was added. A solution of 1.0 equal volume of BIBB (DMG synthesis: 230 mg, DPG: 200 mg, DSG: 180 mg) dissolved in 1.0 mL of dichloromethane was added dropwise to an eggplant flask at room temperature, and the mixture was stirred overnight. An excess amount of hexane was added to the eggplant flask, and the mixture was allowed to stand on ice for 1 hour to precipitate the produced salt and unreacted long-chain alkyl glycerol, and these precipitates were removed by suction filtration. The obtained organic layer was washed with 10 mmol / L hydrochloric acid water (200 mL) and saturated NaCl water (200 mL twice). Anhydrous magnesium sulfate was added to the obtained organic layer, and the mixture was allowed to stand for 1 hour to completely remove water. After removing the precipitate by suction filtration, it was concentrated using an evaporator and allowed to stand under reduced pressure to completely remove the residual solvent. Structural analysis by NMR measurement identified the resulting substances as DMG-Br, DPG-Br and DSG-Br. DMG-Br was white waxy and DPG-Br and DSG-Br were white solids. The yields were all about 60% and the purity was 100%.
The structural formulas of the obtained DMG-Br, DPG-Br and DSG-Br were confirmed by NMR. Table 1 below shows the NMR data of DMG-Br, DPG-Br and DSG-Br.

2. 2. Synthesis of phospholipid polymer having long-chain alkyl group at the end According to the above schemes 2 and 3, activators regenerated by electron transfer (ARGET) type ATRP using the synthesized ATRP initiator is used to add a long-chain alkyl group at the end. A phospholipid polymer (poly (2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) lipid) was synthesized.
MPC as a monomer, DMG-Br, DPG-Br or DSG-Br as an initiator, copper (II) bromide (CuBr ₂ ) as a metal catalyst, and tris (2-pyridylmethyl) amine (TPMA) as a ligand. Ascorbic acid (Asc) was used as a reducing agent. 300 mg of MPC was weighed in a polymerization tube and dissolved in 1.7 mL of a mixed solvent of methanol and 1,4-dioxane (3/2 by volume) to adjust the monomer concentration to 0.5 mol / L. In addition, the polymerization initiators CuBr ₂ , TPMA and Acs were added so as to have a molar ratio of polymerization initiator / CuBr ₂ / TPMA / ascorbic acid = 1 / 0.01 / 0.1 / 1.0. Here, in order to control the degree of polymerization, the molar ratio of MPC to the polymerization initiator was set to 10, 20, 50 and 100. Specifically, the mass of the polymerization initiator is DMG-PMPC10-Br: 66.2 mg, DMG-PMPC20-Br: 33.1 mg, DMG-PMPC50-Br: 13.2 mg, DMG-PMPC100-Br: 6.6 mg, DPG- PMPC10-Br: 71.8 mg, DPG-PMPC20-Br: 35.9 mg, DPG-PMPC50-Br: 14.4 mg, DPG-PMPC100-Br: 7.2 mg, DPG-PMPC10-Br: 77.4 mg, DPG-PMPC20-Br: 38.7 mg, DPG-PMPC50-Br: 15.5 mg, DPG-PMPC100-Br: 7.7 mg, and the mass of CuBr ₂ , TPMA and Acs is determined by the ratio of the monomer to the polymerization initiator regardless of the type of initiator. / When the polymerization initiator is 10, CuBr ₂ : 0.22 mg, TPMA: 2.9 mg, Acs: 17.6 mg, when 20, CuBr ₂ : 0.11 mg, TPMA: 1.5 mg, Acs: 8.8 mg, 50, CuBr ₂ : 0.045 mg, TPMA: 0.58 mg, Acs: 3.5 mg, at 100, CuBr ₂ : 0.022 mg, TPMA: 0.29 mg, Acs: 1.8 mg. Dissolved oxygen was removed by argon bubbling, the mixture was plugged, and the mixture was stirred at 40 ° C. for 24 hours to allow the polymerization reaction to proceed.
The structural formula of each of the obtained PMPC lipids was confirmed by NMR.
Table 2, Table 3 and Table 4 show DMG-PMPC (10, 20, 50, 100), DPG-PMPC (10, 20, 50, 100) and DSG-PMPC (10, 20, 50, 100), respectively. Is shown.

3. 3. Introduction of Fluorescent Molecule to PMPC Lipid Terminal According to the following schemes 4 and 5, a fluorescein molecule was introduced into the terminal of PMPC lipid synthesized by the above method by a click reaction.

Fluorescein (Prop-F) having a propargyl group was synthesized by the method shown below. 118 mg of FITC was weighed in a 50 mL eggplant flask and dissolved in 22 mL of THF. 73 L of propargylamine was added thereto, and the mixture was stirred at room temperature for 24 hours. THF and unreacted propargylamine were removed using an evaporator and these were completely removed under reduced pressure. Structural analysis by NMR measurement identified the obtained orange solid as prop-F. The yield was about 90% and the purity was 100%.
A solution having a concentration of 300 mmol / L was prepared by dissolving 7.8 mg of sodium azide (NaN ₃ ) in 0.4 mL of methanol. The solution immediately after polymerization shown in (1) was added under an argon atmosphere so that the final concentration was 50 mmol / L, the lid was closed again, and the mixture was stirred at 40 ° C. for 24 hours. After removing the precipitated precipitate by centrifugation, the supernatant was reprecipitated with dimethylformamide. The precipitate was washed with diethyl ether and dried under reduced pressure to give a pale yellow solid. By structural analysis by FT-IR measurement, it was identified that the obtained pale yellow solid was a PMPC lipid (N _3- PMPC-lipid) having an azide group at the end.

The N ₃ -PMPC- lipids were weighed 10 mol, it was dissolved in 0.5 mL of methanol containing CuSO ₄ · 5H ₂ O at a concentration of 2.0 mmol / L. To this, 0.4 mL of DMSO solution containing prop-F at a concentration of 25 mmol / L and 0.1 mL of a pure aqueous solution containing sodium ascorbate at a concentration of 100 mmol / L were added in this order, and the mixture was stirred overnight at room temperature. did. The reaction solution was added dropwise to 20 mL of diethyl ether to precipitate the polymer. The supernatant was decanted, thoroughly washed with DMSO, dissolved in pure water, and dialyzed for a predetermined number of days. The obtained solution was freeze-dried to obtain an orange solid. Structural analysis by FT-IR measurement identified the resulting solid as a PMPC lipid with fluorescein at the end.

4. Structural analysis of polymer 2. 100 L of the polymerization solution obtained in 1) was diluted with 500 L of deuterated methanol to prepare a sample for NMR measurement. The NMR measurement was performed 16 times, and the monomer conversion rate was calculated from the ratio of the monomer and the polymer contained in the polymerization solution. Above 2. The molecular weight of the polymer obtained in 1 was estimated by GPC measurement using HFIP containing sodium trifluoroacetic acid at a concentration of 10 mmol / L as an eluent. Poly (methylcrylic) (Shodex, Tokyo, Japan) was used as a standard sample, and HFIP-804 (Shodex, Tokyo, Japan) was used as a column. The flow velocity was 0.5 mL / min and the temperature was 40 ° C.

5. Measurement of Critical Micelle Concentration by DPH (1,6-diphenyl-1,3,5-hexatriene) Method The obtained PMPC lipids (DMG-PMPC, DPG-PMPC, DSG-PMPC) were dissolved in PBS and 10,1 , 0.1, 0.01, 0.001, 0.0001, 0.00001 mg / mL solutions were prepared. DPH was dissolved in THF to prepare a 30 μM solution. This DPH solution was added to and mixed with 1 μL each of PMPC lipid (DMG-PMPC, DPG-PMPC, DSG-PMPC) PBS solution (1 mL), and then incubated at 37 ° C. for 2 hours under shading. Then, the fluorescence intensity of each PMPC lipid solution was measured (FP spectrophotometer (FP8500, Jasco, Tokyo, Japan)) (excitation wavelength: 357 nm, fluorescence wavelength: 430 nm).

6. Micelle particle size measurement
Dissolve PMPC lipids (DMG-PMPC, DPG-PMPC, DSG-PMPC) in PBS to 1 mg / mL and size using Zetasizer nano ZS (Malvern Instruments Co., Ltd., Worcestershire, UK). It was measured. The results are shown in Table 5.

7. Liposomal preparation
DPPC (dipalmitoyl phosphatidylcholine) and cholesterol were used as basic liposomal constituent lipids. PMPC lipids (DPG-PMPC, DSG-PMPC) were added thereto to prepare liposomes modified with PMPC lipids (PMPC-modified liposomes), which were used in the experiment. DPG-PMPC (10, 20, 50, 100) was mixed with DPPC (10 mg) and cholesterol (5.3 mg) at 1.0 mg, 1.9 mg, 4.3 mg and 8.4 mg, respectively, and dissolved in ethanol. .. Then, ethanol was distilled off under reduced pressure with a rotary evaporator. Then, PBS was added so that the total lipid concentration became 10 mg / mL, a stirrer bar was added, and the mixture was stirred at room temperature for 3 hours, and then the liposome size was controlled by an extraction method to prepare liposomes. The extruder was purchased from Avanti and the filter pore diameters used were 0.8 μm, 0.45 μm, 0.22 μm and 0.1 μm (Avanti). Liposomes composed only of unmodified DPPC and cholesterol were also prepared. Further, as PEG-modified liposomes, PEG (5k) -DPPE (1.6 mg) was mixed with DPPC (10 mg) and cholesterol (5.3 mg), and the liposomes were prepared in the same manner.

The particle size of liposomes was measured by the DLS method using Zetasizer nano ZS (Malvern Instruments Co., Ltd., Worcestershire, UK). In addition, it was stored in a PBS solution for 1 month and 3 months (4 ° C.), the particle size was measured, and a long-term stability test was performed (FIGS. 1 to 3).
Immediately after preparation, the liposomes into which the DPG-PMPC lipid was introduced were capable of suppressing aggregation and showed a low PDI (polydispersion index) (similar to PEG-modified liposomes) (Fig. 1). The liposomes into which the DPG-PMPC lipid was introduced did not aggregate even after 1 month, the PDI did not change, and high dispersion stability was maintained. From 1 month to 3 months, PDI increased slightly from 0.1 to 0.2 and the particle size tended to increase slightly, but PMPC-modified liposomes were stably dispersed (similar to PEG-modified liposomes) (Fig.). 3).
Furthermore, liposome preparation and particle size measurement using DSG-PMPC were also performed in the same manner (FIGS. 4 to 6). The preparation is as follows. DSG-PMPC (10, 20, 50, 100) was mixed with DPPC (10 mg) and cholesterol (5.3 mg) at 1.1 mg, 2.1 mg, 4.6 mg and 9.2 mg, respectively, and liposomes were measured. Liposomes were prepared using Extruder as described above (filter pore sizes are 0.8 μm, 0.45 μm, 0.22 μm, 0.1 μm).
In DSG-PMPC10, PDI increased slightly from 0.16 to 0.25 from 1 month to 3 months, and although the particle size increased slightly, it was stably dispersed (about 10%) (about 10%). 5 and 6). In DSG-PMPC20, 50, 100, PDI increased only from 0.10 to 0.14 from 1 month to 3 months, indicating that liposome aggregation did not occur, and the particle size was almost the same. There were (FIGS. 5 and 6).

8. Preparation of Fluorescently Labeled Liposomes and Adsorption Experiments on Substrates
DPPC and cholesterol were used as the basic liposomal constituent lipids. PMPC lipid (DSG-PMPC) was added thereto to prepare liposomes modified with PMPC lipid, which were used in the experiment. DSG-PMPC (10, 20, 50, 100) was mixed with DPPC (10 mg) and cholesterol (5.3 mg) at 1.1 mg, 2.1 mg, 4.6 mg and 9.2 mg, respectively, and dissolved in ethanol. .. Then, ethanol was distilled off under reduced pressure with a rotary evaporator. Unmodified liposomes were also prepared. In addition, as PEG-modified liposomes, DPPC (10 mg) and cholesterol (5.3 mg) were mixed with PEG-DPPE (1.6 mg) to prepare a liposome.
Then, an aqueous solution of carboxyfluorescein (0.5 mM) was added (total lipid concentration: 10 mg / mL), a stirrer bar was added, and the mixture was stirred at room temperature for 3 hours, and then the particle size of the liposome was controlled by the extraction method to control the liposome. Was produced. The filters used are as described in the previous section. This liposome suspension was washed twice by ultracentrifugation (25000 rpm 60 min 4 ° C.) to separate unencapsulated carboxyfluorescein to prepare fluorescently labeled liposomes.
Next, the gold-deposited substrate was immersed in an ethanol solution (1 mM) of 11-amino-1-undecanthiol, hydrochloride (12 hours (room temperature)), washed with ethanol, and self-assembled with amino groups on the surface. A monolayer (NH _2- SAM) was prepared. A liposome suspension (lipid concentration: 5 mg / mL) was added to the substrate, and the mixture was incubated at room temperature for 1 hour. Then, it was washed with PBS and observed with a fluorescence microscope (IX83, Olympus, Tokyo, Japan). The obtained image was analyzed by ImageJ, and the fluorescence intensity was calculated.
As a result, the unmodified liposomes adsorbed to the substrate, whereas the PMPC-modified liposomes suppressed the adsorption of all the samples, which was similar to that of the PEG-modified liposomes. From this result, the coating effect by PMPC was demonstrated.

9. Cell Fluorescein-labeled PMPC lipids were dissolved in phosphate buffered solution (PBS) at a concentration of 5.0 mg / mL. 100 L of CCRF-CEM suspension cultured at a concentration of 6.0 × 10 ⁵ cells / mL was collected in an Eppendorf tube, centrifuged at 150 g, and the supernatant was discarded to recover CCRF-CEM. 50 L of the above polymer solution was added thereto, and the mixture was contacted for 30 minutes. The cell suspension was then centrifuged to discard the supernatant and the precipitated cells were washed by suspending them in 100 L PBS. The state of the cells in the solution was observed with a confocal laser scanning microscope (LSM510, Carl Zeiss Microscopy Co., Ltd., Jena Germany).
As a result, it was confirmed that the cell surface was fluorescently labeled (Fig. 8). Therefore, it was confirmed that the PMPC lipid of the present invention can modify not only artificial lipid membrane structures such as liposomes but also biological membranes such as naturally occurring cell membranes.

The present invention provides a highly biocompatible PMPC-modified membrane structure. Therefore, it can be used for the preparation of liposome preparations and the like, and is expected to be used in the drug discovery and medical fields.

Claims (6)

2-PMPC lipid formed by binding a polymer consisting of methacryloyloxyethyl phosphorylcholine (MPC). The PMPC lipid according to claim 1, which is obtained by binding poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) of the following formula (1).
(In formula (1), X is a functional group and n is an integer of 10 or more) A lipid membrane structure containing the PMPC lipid according to claim 1 or 2 as a constituent lipid. The lipid membrane structure according to claim 3, which is a liposome. The lipid membrane structure according to claim 3, wherein the lipid membrane structure is a biological membrane. The lipid membrane structure modifier containing the PMPC lipid according to claim 1 or 2.