Polysarcosine and its uses

Abstract

To provide a coacervate that can be used without any problem as a carrier in a drug delivery system, and a method for producing the novel coacervate.SOLUTION: A polymer has oligosaccharide chains at its termini and includes a constitutional unit derived from N-methyl glycine as a repeat unit.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to polysarcosine and its uses. [Background technology]

[0002] A drug delivery system (DDS) is a system that controls the distribution of drugs within the body quantitatively, spatially, and temporally. To function as a carrier in a drug delivery system, it must meet certain requirements, including being of controlled size (30 nm to 200 nm), not being damaged in the biological environment, being able to efficiently encapsulate drugs, and having biocompatibility in itself and its degradation products. To date, membrane-bound capsules and membrane-free (coacervate) types are known to be used as carriers for drug delivery systems and as in vivo nanoreactors. Capsule-type formulations allow for the encapsulation of drugs or other substances within a capsule formed by phospholipids, polymers, etc. For example, liposome formulations containing anticancer drugs are well-known. Coacervates are polymers that can support drugs or proteins in droplets in water. For example, ionic coacervates described in Patent Documents 1 and 2 are known. Polyionic complex coacervates are complexes produced by electrostatic interactions when linear polymers with opposite charges are reacted in an aqueous solution.

[0003] However, these ionic coacervates are unstable in aqueous solutions containing salts, as is the case in living organisms, making them difficult to use as carriers in drug delivery systems, and improvements in their properties are needed. As for non-ionic nano-sized coacervates that are substantially charge-free, only a few peptides with specific repeating sequences are known (Patent Document 3). [Prior art documents] [Patent Documents]

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a coacervate that has no problem in use as a carrier in a drug delivery system.

Means for Solving the Problems

[0006] The present inventors have intensively studied to solve the above problems. In the process of the study, the present inventors succeeded in synthesizing a novel polymer that is nonionic, that is, substantially uncharged. The present inventors have further found that the polymer easily forms molecular aggregates in water and a desired coacervate can be obtained. The present invention has been completed by further improving based on such findings. This disclosure includes, for example, the subject matter described in the following items. Item 1. A polymer having an oligosaccharide chain at the terminal and having a structural unit derived from N-methylglycine as a repeating unit. Item 2. The polymer according to Item 1, wherein the number average molecular weight of the polymer is 700 to 35000. Item 3. The polymer according to Item 1 or 2, which is substantially nonionic. Item 4. [[ID=...]] Item 5. The coacervate according to Item 4, having an average particle diameter of 50 nm to 200 nm. Item 6. ​A coacervate according to item 4 or 5, characterized by being stable in a saline solution. Section 7. A method for producing a coacervate, comprising the step (1) of dissolving in water a polymer having oligosaccharide chains at its termini and having repeating units derived from N-methylglycine as structural units, in order to obtain a coacervate. Section 8. The manufacturing method according to item 7, further comprising the step (2) of ultrasonically treating the coacervate. [Effects of the Invention]

[0007] Homopolysarcosine (hereinafter simply referred to as "polysarcosine") is soluble in water and therefore cannot undergo self-association in water, thus failing to form aggregates. In contrast, the polysarcosine of the present invention, in which an oligosaccharide chain is attached to the end of the polysarcosine molecule, undergoes self-association and coacervation, enabling the formation of monodisperse nanocolloids in water. The phenomenon of coacervation occurring by introducing an oligosaccharide chain to the end of polysarcosine (glycosylation of peptoids) is a surprising finding that even the inventors could not have predicted.

[0008] Polysarcosine is a water-soluble, nonionic peptide derivative that possesses properties suitable for biomedical applications, including biocompatibility, biodegradability, and stealth capabilities within the immune system. Therefore, the novel polymer of the present invention, glycosylated polysarcosine, can also be used for biomedical applications without any problems. Even more remarkably, the coacervates formed by the polysarcosine into which the sugar chains of the present invention are introduced possess the excellent property of being stable even in aqueous solutions where salts are present at high concentrations. [Brief explanation of the drawing]

[0009] [Figure 1] This is the molecular structure of maltooligotose-polysarcosine. [Figure 2]This is a schematic diagram of a coacervate formed from maltooligotose-polysarcosine. [Modes for carrying out the invention]

[0010] The embodiments of the present invention will be described in more detail below. The present invention preferably includes, but is not limited to, oligosaccharide-modified polysarcosines, and encompasses all that is disclosed herein and recognizable to those skilled in the art.

[0011] 1. Polymer of the present invention The polymer of the present invention is a polymer (polysarcosine) having oligosaccharide chains at its terminals and using N-methylglycine-derived structural units as repeating units.

[0012] In the polymer of the present invention, the oligosaccharide chain consists of 3 to 8 monosaccharides. The oligosaccharide of the present invention is not particularly limited as long as it is a known oligosaccharide. There are no particular restrictions on the monosaccharides that make up the oligosaccharide, and examples include glucose, fructose, mannose, galactose, ribose, etc. Specifically, examples of oligosaccharides include maltotriose, maltotetraose, maltopentaose, maltoheptaose, etc. Among these, maltotriose, maltotetraose, or maltopentaose are preferred.

[0013] In the polymer of the present invention, the number of repeating units (n) derived from N-methylglycine is usually 10 to 500. The lower limit of this range may be, for example, 10, 15, or 20. The upper limit of this range may be, for example, 500, 400, 300, 200, 150, or 100. The number of repeating units derived from N-methylglycine is particularly preferably 20 to 100.

[0014] In the polymer of the present invention, the oligosaccharide chain and the polysarcosine, which has repeating units derived from N-methylglycine, may be directly bonded or bonded via a linker. The type of linker is not particularly limited as long as the self-assembly ability of the polymer of the present invention is maintained. In the present invention, "self-assembly ability" refers to the property of being able to spontaneously form coacervates when dissolved in water.

[0015] The number-average molecular weight of the polymer of the present invention is typically 700 to 40000. The lower limit of this range may be, for example, 700, 800, 900, 1000, 1100, 1200, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, or 3000. The upper limit of this range may be, for example, 35000, 30000, 25000, 20000, 15000, 10000, 9000, 8000, or 12000. The number-average molecular weight of the polymer of the present invention is particularly preferably 1400 to 12000.

[0016] The number-average molecular weight is usually measured by known methods. For example, as a specific method, the number-average molecular weight can be determined as the converted mass of PMMA, PEG, etc., using the GPC (Gel Permeation Chromatography) method described later.

[0017] The polymer of the present invention is produced, for example, according to the following production methods A and B. [Manufacturing method A] The polymer of the present invention is produced by reacting an oligosaccharide chain with homopolysarcosine. The starting material, the oligosaccharide chain, is either known or can be easily produced by known methods. The other starting material, homopolysarcosine, can be easily produced by polymerizing N-methylglycine (sarcosine) by known methods. These manufacturing conditions are as described in Manufacturing Example 1 below, and the desired target compound can be produced by referring to the manufacturing conditions described in Manufacturing Example 1. [Manufacturing method B] The polymer of the present invention is produced by polymerizing N-methylglycine (sarcosine) to the terminal end of an oligosaccharide chain. The polymerization reaction conditions are as described in Production Example 2 below, and the desired target compound can be produced by referring to the production conditions described in Example 2.

[0018] The polymers of the present invention obtained by the above-described manufacturing methods A and B can be easily isolated and purified from the reaction mixture by appropriately employing known isolation and purification methods, such as distillation, centrifugation, solvent extraction, recrystallization, gel filtration, or silica gel chromatography.

[0019] The polymer of the present invention spontaneously forms micrometer-sized coacervates when dissolved in water.

[0020] 2. The coacervate of the present invention The coacervate of the present invention includes a polymer having oligosaccharide chains at its termini and consisting of repeating units derived from N-methylglycine.

[0021] In this invention, the term "coacervate" refers to a droplet containing colloidal particles and includes nano-sized coacervates, microspheres, and nanospheres that have been miniaturized by ultrasonic treatment. Here, microspheres refer to spherical formulations with a particle diameter of several μm, and nanospheres refer to spherical formulations with a particle diameter of less than 1 μm.

[0022] The coacervate of the present invention may contain, in addition to water, any acceptable additives as needed. Examples of water include tap water, deionized water, distilled water, pure water, ultrapure water, and sterile water.

[0023] When the coacervate of the present invention is used as a pharmaceutical, it can be formulated by methods known to those skilled in the art. In addition to water, the coacervate can be formed with any pharmaceutically acceptable liquid, for example, as needed, and the drug can be encapsulated in it to prepare it in a form that can be used as a carrier in a drug delivery system. The term "pharmaceutically acceptable liquid" broadly includes known substances that are inert and used as diluents or vehicles for drugs.

[0024] The coacervate of the present invention is spontaneously formed by dissolving the polymer of the present invention in water. The coacervate of the present invention is further refined by ultrasonic treatment to form nano-sized coacervates.

[0025] In forming the coacervate of the present invention, the ratio of the polymer of the present invention to water is not particularly limited as long as the desired coacervate is obtained, and is, for example, 0.01% by weight or more and less than 100% by weight. The lower limit of this range may be, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The upper limit of this range may be, for example, 99, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10. The ratio of the polymer of the present invention to water is preferably 0.01% to 10% by weight, and more preferably 1% to 10% by weight.

[0026] In this invention, the conditions for ultrasonic treatment are not particularly limited as long as the desired nano-sized coacervate can be obtained. In this invention, the ultrasonic treatment temperature is not particularly limited as long as the desired nano-sized coacervate can be obtained, and can be, for example, 4°C to 40°C. In addition, in this invention, the ultrasonic oscillation frequency is not particularly limited as long as the desired nano-sized coacervate can be obtained, and can be, for example, 20kHz to 100kHz. Furthermore, in this invention, the ultrasonic treatment time is not particularly limited as long as the desired nano-sized coacervate can be obtained, and can be, for example, 1 minute to 60 minutes. In this invention, the apparatus used for ultrasonic treatment is not particularly limited as long as the desired nano-sized coacervate can be obtained, and can be, for example, a bath-type sonicator, a homogenizer, etc.

[0027] The average particle size of the coacervate of the present invention is typically 50 to 200 nm. Preferably, it is 50 to 150 nm. More preferably, it is 50 to 100 nm.

[0028] The average particle size of the coacervate of the present invention can be measured by dynamic light scattering (DLS).

[0029] The coacervate of the present invention is characterized by being substantially nonionic and stable in aqueous saline solutions.

[0030] 3. The method for producing coacervate according to the present invention The present invention provides a method for producing coacervate, comprising step (1) dissolving in water a polymer having oligosaccharide chains at its termini and consisting of repeating units derived from N-methylglycine, in order to obtain a coacervate.

[0031] The method for producing coacervates of the present invention may further include a step (2) of ultrasonically treating the self-assembled polymer. This step (2) can form finely divided coacervates.

[0032] The average particle size of the miniaturized coacervate of the present invention is typically 10 to 100 nm. Preferably, it is 20 to 100 nm. More preferably, it is 20 to 50 nm.

[0033] The average particle size of the miniaturized coacervate of the present invention can be measured by dynamic light scattering (DLS).

[0034] In this specification, the term "comprising" includes both "consisting essentially of" and "consisting of." Furthermore, this disclosure encompasses all any combination of the constituent elements described herein. Moreover, in this invention, "A to B (where A and B represent any numerical values)" is synonymous with "A or greater and B or less."

[0035] This disclosure encompasses all subject matter consisting of any combination of each of the combinatable properties described herein. [Examples]

[0036] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0037] The following raw materials were used in this example. • Maltopentaose (purity > 98%) • Maltotriose (purity > 98%) • Acetic anhydride (purity > 93%) • Ammonium molybdate (purity > 99.98%) ·Copper(I) bromide (purity > 95%) • Propargylamine (purity > 95%) • Sarcosine (purity > 98%) Methyl chloroformate (purity > 96%) • Thionyl chloride (purity > 98%) • Benzylamine (purity > 99%) • Azidoacetic acid (purity > 97%) • Pyrene (purity > 97%) • Fluorescein isothiocyanate (purity > 97%) Rhodamine B • Fluorescein 1,8-ANS (purity > 98%) Maltopentaose, maltotriose, acetic anhydride, and ammonium molybdate and copper(I) bromide were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Propargylamine, sarcosine, methyl chloroformate, thionyl chloride, benzylamine, azidoacetic acid, pyrene, and fluorescein isothiocyanate were purchased from Tokyo Chemical Industries, Ltd. Rhodamine B and fluorescein were purchased from Sigma-Aldrich Co., LLC. 1,8-ANS was purchased from Cayman Chemical Company.

[0038] The following method was used in this embodiment. DLS measurement DLS measurements were performed using a Zetasizer Nano ZS instrument (Malvern Instruments) operating at a wavelength of 632.8 nm and a detection angle of 173°. 1 H NMR measurement All starting materials and solvents were used without further purification. The nuclear magnetic resonance spectra were obtained. 1 To obtain the H-NMR spectrum, a Bruker Avance III 400 MHz spectrometer was used with methanol-d4. The chemical shift (δ) is expressed as part per million. 1 The 1H-NMR spectrum is reported by comparison with the solvent peak as an internal standard. The mass spectrum was recorded using a Thermo Exactive spectrometer. GPC (Gel Permeation Chromatography) method The molecular weight distribution (MWD) curve, number-average molecular weight (Mn), and polydispersity index (DM) were obtained using a linear type polystyrene gel column (TSKgel SuperHM-M, manufactured by Tosoh Techno Systems Co., Ltd.; exclusion limit: 4 × 10⁶ g mol). -1Particle size: 3 μm; Pore size: N / A; 6.0 mmi.d. × 15 cm), using an instrument equipped with an HLC-8320GPC (manufactured by Tosoh Techno Systems Co., Ltd.), refractive index, and UV-Vis detector (λ = 254 nm), in N,N-dimethylformamide (DMF) containing 10 mM LiBr at 40°C (flow rate = 0.50 mL min). -1 The measurement was performed by GPC. The column was calibrated against 12 different polymethylene methacrylates (PMMAs) with varying number-average molecular weights as molecular weight standards [PSS:Mp = 8.00 × 10⁻¹⁰]. 2 ~2.20×10 6 g mol -1 ; DM(GPC) = 1.04-1.22].

[0039] Examples of polymer production [Production Example 1: Synthesis of Maltooligotose-Polysarcosine (Production Method A)] (1) Synthesis of polysarcosine segments Polysarcosine segments of different lengths (Sar-10.7, Sar-8.0, Sar-6.1, Sar-4.5, Sar-2.6) were synthesized (Scheme 1).

[0040] [Compound [4]: ​​Synthesis of N-(methoxycarbonyl)-N-methylglycine] Sarcosine (30.0 g, 337 mmol) was dissolved in 337 mL of 1 M NaOH aqueous solution, and methyl chloroformate (33.9 mL, 438 mmol) was added dropwise while cooling in an ice bath at 0°C. The mixture was stirred at room temperature for 6 hours. Next, the pH of the reaction solution was adjusted from 11 to 6 with 2 N HCl aqueous solution. After extracting the compound with diethyl ether, the solvent was removed under reduced pressure. Compound [4] was a white solid (42.54 g, 288 mmol, 84%).

[0041] [Compound [5]: Synthesis of sarcosine-N-carboxyanhydride (NCA)] Compound [4] (5.00 g, 34.0 mmol) was dissolved in SOCl2 (50 mL, 0.693 mol), and the solution was stirred in an oil bath at 70°C for 12 minutes. NCA was precipitated using petroleum ether. The crystals were collected by filtration and dried under vacuum to obtain compound [5] (1.28 g, 11.12 mmol, 33%).

[0042] [Compounds [1-1]~[1-5]: Synthesis of polysarcosine homopolymers] Compound [5] (1.16 g, 10.1 mmol) was dissolved in dry dimethylformamide (10 mL), and benzylamine (10.8 μL, 101 μmol) was added to the solution. The mixture was stirred under an argon atmosphere at room temperature for 12 hours. Next, the polymer solution was added to diethyl ether and reprecipitation was performed. The amount of compound [5] was adjusted to 0.928 g (8.08 mmol) in 8 mL of dry DMF, 0.696 g (6.06 mmol) in 6 mL of dry DMF, 0.464 g (4.04 mmol) in 4 mL of dry DMF, and 0.232 g (2.02 mmol) in 4 mL of dry DMF. The ratio of compound [5] to benzylamine was adjusted to obtain polysarcosine of different lengths. In the preparation of polysarcosine homopolymers of all lengths, the resulting solid was a white powder.

[0043] [Synthesis of azide-functionalized polysarcosine homopolymers [Sar-10.7]~[Sar-2.6]] Compound [1-1] (100 mg, 13.9 μmol) was dissolved in 3 mL of dry DMF. (1-Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) (25.2 mg, 58.9 μmol), ethyl cyano(hydroxyimino)acetate (Oxyma) (8.39 mg, 58.9 μmol), and 2-azidoacetic acid (10 μL, 134 μmol) were then added to the solution, followed by N,N-di(isopropyl)(ethyl)amine (10 μL, 101 μmol). The mixture was stirred at room temperature for 24 h. The solvent was removed under reduced pressure, and the residue was washed with ethyl acetate and diethyl ether. The resulting solid was a slightly yellow powder (90 mg, 12.3 μmol, 88%). Compound [1-2], [1-3], [1-4] or [1-5] was used, and the amounts of COMU, Oxyma and 2-azidoacetic acid were adjusted appropriately to produce the respective target compounds.

[0044]

Chemical Structure

[0045] (2) Synthesis of oligosaccharide chains Maltotriose and maltopentaose were synthesized (Scheme 2).

[0046] [Compound [S3]: Alkyne-functionalized maltotriose] Maltotriose (Compound [8], 1.09 g, 2.16 mmol) was dissolved in propargylamine (2.6 mL, 40.6 mmol), and the mixture was stirred at room temperature for 144 h. Next, the solution was dropped into diethyl ether, and the resulting white solid was separated by filtration. The white solid was dissolved in a mixture of dry methanol (28 mL) and acetic anhydride (15.0 mL, 159 mmol). The solution was stirred at room temperature for 144 h. The solvent was removed under reduced pressure, and the residue was dried in vacuo. The resulting solid was a white powder (993 mg, 90%). For Compound [S3] 1The results of the 1H NMR measurement are as follows: 1 ¹H NMR (400 MHz, methanol-d4): δ = 2.22 and 2.28, 2.49 and 2.74, 3.40-4.16, 5.06-5.10, 4.81 and 5.43.

[0047] [Compound [S5]: Alkyne-functionalized maltopentaose] Maltopentaose (compound [9], 1.09 g, 1.31 mmol) was dissolved in propargylamine (2.6 mL, 40.6 mmol), and the mixture was stirred at room temperature for 144 hours. Next, the solution was added dropwise to diethyl ether, and the resulting white solid was separated by filtration. The white solid was dissolved in a mixture of dry methanol (280 mL) and acetic anhydride (15.0 mL, 159 mmol). The solution was stirred at room temperature for 144 hours. The solvent was removed under reduced pressure, and the residue was dried in vacuum. The resulting solid was a white powder (1.06 g, 89%). Compound [S5] 1 The results of the 1H NMR measurement are as follows: 1 ¹H NMR (400 MHz, methanol-d4): δ = 2.22 and 2.28, 2.49 and 2.77, 3.4-4.16, 5.15-5.20, 4.91 and 5.53.

[0048] [ka]

[0049] (3) Synthesis of maltooligotose-polysarcosine [Polymer [S3-Sar10.7] to [S3-Sar2.6]: Maltotriose-β-Polysarcosine] Polysarcosine segments of different lengths synthesized in Scheme 1 were combined with maltotriose synthesized in Scheme 2 (Scheme 3). Compound [S3] (30.0 mg, 51.4 μmol) and compound [Sar-10.7] (50.0 mg, 7.93 μmol) were dissolved in 2 mL of dry DMF. CuBr(I) (2.0 mg, 13.9 μmol) was added to the solution, and the solution was stirred at room temperature for 72 hours. Next, the solution was passed through SiliaMetS® triamine (manufactured by SiliCycle) to remove copper. The solvent was evaporated, and the residue was purified by gel filtration chromatography using Sephadex LH-20 (eluent: methanol). Maltotriose-β-polysarcosines of other lengths (from S3-Sar8.0 to S3-Sar2.6) were synthesized using the same procedure. All block copolymers were slightly yellow solids. The results of analysis of the polymers obtained by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) are shown below. m / z:[S3-Sar10.7+Na] + Calculated value C 404 H 667 N 129 NaO 141 For example, if the value is 9584.91, the measured value is 9584.62. m / z:[S3-Sar8.0+Na] + Calculated value C 293 H 482 N 92 NaO 104 For example, if the value is 6955.53, the actual measured value is 6955.62. m / z:[S3-Sar6.1+Na] + Calculated value C 227 C 372 N 70 NaO 82 For example, if the value is 5391.71, the measured value is 5392.12. m / z:[S3-Sar4.5+Na] + Calculated value C 134 H 217 N 39 NaO 51 For example, if the value is 3190.57, the measured value is 3189.72. m / z:[S3-Sar2.6+Na] + Calculated value C 98 H157 BN 27 NaO 39 For example, in the case of 2336.11, the measured value is 2335.99.

[0050] [ka]

[0051] [Polymer [S5-Sar10.7] to [S5-Sar2.6]: Maltopentaose-β-polysarcosine] Polysarcosine segments of different lengths synthesized in Scheme 1 were combined with maltopentaose synthesized in Scheme 2 (Scheme 4). Compound [S5] (40.0 mg, 44.1 μmol) and compound [Sar-10.7] (50.0 mg, 7.93 μmol) were dissolved in 2 mL of dry DMF. CuBr(I) (2.0 mg, 13.9 μmol) was added to the solution, and the solution was stirred at room temperature for 72 hours. Next, the solution was passed through SiliaMetS® triamine (SiliCycle) to remove copper. The solvent was evaporated, and the residue was purified by gel filtration chromatography using Sephadex LH-20 (eluent: methanol). Maltopentaose-β-polysarcosines of other lengths (from S5-Sar8.0 to S5-Sar2.6) were synthesized using the same procedure. All block copolymers were slightly yellowish solids. MALDI-TOF (Matrix: CHCA) m / z:[S5-Sar10.7+Na] + Calculated value C 455 H 752 N 142 NaO 164 For example, if the value is 10834.50, the actual measured value is 10832.90. m / z:[S5-Sar8.0+Na] + Calculated value C 293 H 482 N 88 NaO 110 For example, if the value is 6996.96, the actual measured value is 6997.62. m / z:[S5-Sar6.1+Na] + Calculated value C 233 H 382 N 68 NaO 90 For example, if the value is 5574.75, the actual measured value is 5575.62. m / z:[S5-Sar4.5+Na] + Calculated value C 173 H 282 N 48 NaO 70 For example, if the value is 4154.00, the measured value is 4154.02. m / z:[S5-Sar2.6+Na] + Calculated value C 110 H 177 N 27 NaO 49 For example, if the value is 2661.22, the measured value is 2661.35.

[0052] [ka]

[0053] [Production Example 2: Synthesis of Maltooligotose-Polysarcosine (Production Method B)] (1) Addition of Boc-ethylenediamine to maltotriose Maltotriose (500 mg, 0.99 mmol) was mixed with Boc-ethylenediamine (10.0 mL, 63.1 mmol) and stirred at 40°C for 72 hours. The resulting viscous liquid was diluted with methanol (30 mL) and then reprecipitation was performed by adding it dropwise to diethyl ether to obtain a pale yellow precipitate. The precipitate was filtered off and then vacuum dried. The resulting pale yellow solid was a powder (583 mg, 85%).

[0054] (2) De-Boc The powder obtained by the above method was deprotected (Boc removed) by stirring in a 4N HCl aqueous solution at 25°C for 3 hours. After stirring, the hydrochloric acid aqueous solution and the Boc removal by-products in the clear aqueous solution were removed by vacuum distillation to obtain a pale yellow solid.

[0055] (3) Sarcosine elongation Sar-NCA was prepared from Moc-Sar-OH by the same method as in Preparation Example 1. Sar-NCA (100 mg, 0.87 mmol) was dissolved in super-dehydrated DMF (7.7 mL). Maltotriose-ethylenediamine (5.1 mg, 0.0087 mmol) prepared by the method described above was dissolved in 1 mL of super-dehydrated DMF and added to the previously prepared Sar-NCA solution in DMF, and the mixture was stirred for 24 hours. The DMF was then removed by vacuum distillation to obtain a pale yellow solid. The obtained solid was dissolved again in methanol and re-precipitated and purified using diethyl ether as a poor solvent to obtain a pale yellow solid.

[0056] Evaluation of oligosaccharide-added polysarcosines from production examples 1 and 2 [Manufacturing example 1 (manufacturing method A)] (1) Synthesis of oligosaccharide-added polysarcosine To investigate the effect of oligosaccharide and polysarcosine length on self-assembly, maltooligotose-polysarcosines (hereinafter referred to as oligosaccharide-added polysarcosines) with 20, 40, 60, 80, or 100 sarcosine units were designed with trisaccharides or pentasaccharides (Figure 1). Benzylamine was injected as an initiator into 1000 mM sarcosine NCA in anhydrous DMF at [benzylamine]:[NCA] = 10.0:1000, 12.5:1000, 16.7:1000, 25.0:1000, or 50.0:1000. The solutions were stirred at 20°C for 12 hours under an argon atmosphere in millimoles-to-millimoles basis. Gel permeation chromatograms (GPCs) of all obtained polymers (compounds Sar-10.7 to Sar-2.6) showed a single peak with an extremely narrow molecular weight distribution, and the peak top with a strong molecular weight reflected the preparation molar ratio of the initiator to sarcosine NCA (Table 1). Table 1 shows the results of the number-average molecular weight measurement of polysarcosine segments by the GPC method. [Table 1]

[0057] The N-terminus of polysarcosine was capped with azidoacetic acid. The resulting azido-functionalized polysarcosine segment was coupled to alkyne-functionalized maltotriose (S3) or maltopentaose (S5) via a Cu-catalyzed azido-alkyne cycloaddition reaction (CuAAc). Gel permeation chromatography (GPC) of all the resulting polymers (compounds S5-Sar10.7~S5-Sar2.6 and S3-Sar10.7~S3-Sar2.6) also showed a single peak with a very narrow molecular weight distribution (Table 2). Similarly to the above, the results of measuring the number-average molecular weight of maltooligotose-polysarcosine by GPC method, 1 Table 2 shows the degree of polymerization (DP) determined by 1H NMR. [Table 2] Table 2 also shows the DP of each glycosylated polysarcosine, consisting of molecular weights calculated from GPC using PMMA as the molecular weight standard. The synthesis of these polymers was confirmed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Based on these observations, we confirmed that the desired glycosylated polysarcosines, namely maltotriose-polysarcosine or maltopentaose-polysarcosine, were synthesized.

[0058] (2) Self-assembly of oligosaccharide-added polysarcosine First, we evaluated the size of self-assembled oligosaccharide-modified polysarcosines in aqueous solutions using dynamic light scattering (DLS) (Table 3). Table 3 shows the size, distribution, and scattering intensity (n = 5) of self-assembled oligosaccharide-modified polysarcosine, measured by DLS at 20°C. The sonication time was 5 minutes, and the solution concentration was 10.0 mg / mL. [Table 3] A methanol solution of oligosaccharide-modified polysarcosine was added to a glass tube, and the solvent was removed under reduced pressure. The resulting polymer thin film was hydrated with ultrapure water to obtain an aqueous polymer solution with a concentration of 10.0 mg / mL. All oligosaccharide-modified polysarcosine dissolved immediately in water. The aqueous solution of oligosaccharide-modified polysarcosine was sonicated for 5 minutes in a bath-type sonicator at 20°C (40 kHz, 130 W). In the case of maltotriose-polysarcosine (S3-Sar series), monodisperse particles with diameters of 150-220 nm were observed, and the size was independent of the molecular weight of the polysarcosine (Table 3). Conversely, the size of particles formed from maltopentaose-polysarcosine (S5-Sar series) varied depending on the molecular weight of the polysarcosine. The high molecular weight S5-Sar series (S5-Sar10.7 and S5-Sar8.0) formed monodisperse particles in the 150–220 nm range, similar to the S3-Sar series. Other S5-Sar compounds with lower molecular weight polysarcosine (S5-Sar6.1, S5-Sar4.5, and S5-Sar2.6) formed larger particles over a relatively wide dispersion range (Table 3).

[0059] [Manufacturing example 2 (manufacturing method B)] The obtained polymer was subjected to GPC analysis using the same method as described in Production Example 1. The weight-average molecular weight in PMMA terms was 10700, the number-average molecular weight was 8800, and the dispersion index Mw / Mn was 1.24. The obtained polymer was dissolved in ultrapure water at a concentration of 1.0 mg / mL, and then subjected to sonication for 5 minutes using the same apparatus as in Production Example 1. DLS measurement was then performed, and the particle size was found to be 308 nm.

[0060] The schematic diagram of the coacervate obtained above is shown in Figure 2. [Industrial applicability]

[0061] Nanoparticles with particle sizes controlled to tens to hundreds of nanometers are known to have the property of specifically accumulating in solid tumors, for example, and the polymers and coacervates of the present invention are promising materials with potential for medical applications. Furthermore, both the oligosaccharides and peptoids used in the present invention are biocompatible materials and are stable even in environments where salts in living organisms are present, so they can be used, for example, as base materials for drug delivery systems and in vivo nanoreactors.

Claims

1. A coacervate containing a polymer having oligosaccharide chains at its termini and repeating units derived from N-methylglycine.

2. The coacervate according to claim 1, wherein the average particle diameter is 50 nm to 200 nm.

3. A method for producing a coacervate, comprising the step of dissolving in water a polymer having oligosaccharide chains at its termini and consisting of repeating units derived from N-methylglycine, to obtain a coacervate.

4. The manufacturing method according to claim 3, further comprising the step of ultrasonically treating the coacervate.

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