KR102567268B1 - A pH-sensitive amphiphilic block copolymer, a manufacturing method thereof, a polymer micelle comprising the pH-sensitive amphiphilic block copolymer - Google Patents

Abstract

The present invention relates to a pH-sensitive amphiphilic block copolymer, a method for preparing the same, and a polymeric micelle containing the pH-sensitive amphiphilic block copolymer, and more particularly, polycyclohexyl having hydrophobicity at one end of a hydrophilic polymer. By copolymerizing oxyethyl glycidyl ether (PCHGE), it is possible to form self-assembled polymeric micelles that can be encapsulated by strong intermolecular hydrophobic interactions of CHGE, and has an advantage of excellent human body stability. In addition, the polymeric micelles using this have excellent encapsulation stability and biocompatibility, and due to the acetal bond that reacts to the pH of CHGE, the pH is adjusted or the number of CHGE monomers is controlled to control the decomposition rate of the micelles so that the drug can be effectively released. can be adjusted

Description

A pH-sensitive amphiphilic block copolymer, a manufacturing method thereof, a polymer micelle comprising the pH-sensitive amphiphilic block copolymer block copolymer}

The present invention relates to a pH-sensitive amphiphilic block copolymer, a method for preparing the same, and a polymeric micelle containing the pH-sensitive amphiphilic block copolymer.

Chemotherapy is the most common means of treating cancer. However, it is very important to develop new drug delivery systems with improved efficiency to overcome the inherent limitations of therapeutics such as high toxicity, rapid elimination and tumor selectivity. In order to solve this problem, research and development on polymeric drug carriers including polymeric drug conjugates, liposomes, and polymeric micelles have been conducted.

Among these, polymeric micelles have been widely used and studied as ideal drug delivery carriers with unique advantages such as highly tunable loading efficiency and capacity, facile deformation, long circulation time in the circulatory system, release side effects, and tunable dissolution kinetics. Although these polymeric micelles have made great progress for efficient drug delivery, the stability problem of applying them is a task to be solved.

The stability of drug carriers in the biological environment is very important. Adsorption of various protein components in contact with biological mediators negatively affects the stability of the vehicle and becomes a factor in reducing the effectiveness of drug delivery. In addition, the stability of polymeric micelles in the bloodstream is reduced due to dynamic equilibrium shifts. In order to solve these problems, the stability of polymeric micelles can be improved when non-covalent interactions such as hydrophobicity, electrostatics, host-guest, hydrogen bonds, stereocomplexes, and coordination interactions are introduced into polymeric micelles.

In the existing literature, disulfide-linked deoxycholic acid-hyaluronic acid, which is a polymeric micelle having a reduction reaction, was synthesized to improve the hydrophobicity of the amphiphilic copolymer. The polymeric micelles were degraded in the presence of glutathione and exhibited accelerated hydrophobic drug release. The stearate-g-dextran graft copolymers synthesized in another literature have different grafting degrees of hydrophobic stearate, and as the ratio of the hydrophobic stearate increases, the loading capacity and encapsulation efficiency increase and the size decreases. Encapsulation of hydrophobic molecules in amphiphilic assemblies is also related to hydrophobicity. Accordingly, increasing the hydrophobicity of self-assembled micellar cores can improve interactions with hydrophobic therapeutics and improve loading capacity and efficiency.

In addition, sophisticated delivery is another important element of DDS. Polymeric micelles with specific chemical functionalities that respond to various endogenous stimuli such as pH, redox, enzymes or proteins and nucleic acids have been developed to deliver therapeutic compounds and control drug release at disease sites. Due to the acidic microenvironment of cancer cells, pH-responsive micelles with acid labile bonds such as ketals, acetals, hydrazones and orthoesters were used.

Synthesis of a series of pH-responsive glycidyl ethers with acetal groups, including tetrahydrofuranyl glycidyl ether (TFGE) and tetrahydropyranyl glycidyl ether (TPGE) as new functional epoxide monomers for drug delivery did However, polymeric micelles based on TFGE and TPGE monomers have been successful as drug carriers, but achieving high doses and human body stability to reduce side effects of therapeutics remains a challenge.

Korean Patent Registration No. 10-2187144

In order to solve the above problems, an object of the present invention is to provide a pH-sensitive amphiphilic block copolymer capable of forming self-assembled micelles.

In addition, an object of the present invention is to provide a polymeric micelle having excellent encapsulation stability and biocompatibility, including the pH-sensitive amphiphilic block copolymer.

Another object of the present invention is to provide a pH-sensitive targeting delivery system comprising the polymeric micelles.

Another object of the present invention is to provide a method for preparing a pH-sensitive amphiphilic block copolymer.

The present invention provides a pH-sensitive amphiphilic block copolymer in which polycyclohexyloxyethyl glycidyl ether (PCHGE) having hydrophobicity is copolymerized by anionic ring-opening polymerization at one end of a hydrophilic polymer.

In addition, the present invention is the pH-sensitive amphiphilic block copolymer; and a molecular imaging marker, a contrast agent, or a therapeutic agent bonded to polycyclohexyloxyethyl glycidyl ether (PCHGE) of the pH-sensitive amphiphilic block copolymer.

In addition, the present invention provides a pH-sensitive targeting delivery system comprising the polymeric micelles.

In addition, the present invention comprises the steps of synthesizing cyclohexyloxyethyl glycidyl ether (CHGE); and preparing a pH-sensitive amphiphilic block copolymer by adding a hydrophilic polymer, the cyclohexyloxyethyl glycidyl ether (CHGE), and a catalyst to an organic solvent and conducting anionic ring-opening polymerization. A method for producing a block copolymer is provided.

The pH-sensitive amphiphilic block copolymer according to the present invention is capable of encapsulation by copolymerizing polycyclohexyloxyethyl glycidyl ether (PCHGE) having hydrophobicity at one end of a hydrophilic polymer due to strong intermolecular hydrophobic interactions of CHGE. It can form self-assembled polymeric micelles, and has the advantage of excellent human body stability.

In addition, the polymeric micelles according to the present invention have excellent encapsulation stability and biocompatibility, and can effectively release drugs by controlling the decomposition rate of micelles by controlling the pH or the number of CHGE monomers due to the acetal bond that reacts to the pH of CHGE. can be adjusted so that

The effects of the present invention are not limited to the effects mentioned above. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

1 is a 1H NMR spectrum analysis result of CHGE monomer (a), PCHGE homopolymer (b), and mPEG-b-PCHGE block copolymer (c) prepared in Example 1 and Comparative Example 1 of the present invention.
Figure 2 shows the results of MALDI-ToF mass spectrum analysis from 2750 to 4500 Da for the mPEG-b-PCHGE block copolymer prepared in Example 1 of the present invention.
Figure 3 schematically shows the energy diagram (a) of the hydrolysis reaction coordinates of various model acetal side chain compounds (CHPE, TPPE, TFPE, EEPE) and the proposed hydrolysis mechanism (b) of CHPE.
4 is an AFM image (df) of Cy1, Cy2, and Cy3 micelles prepared in Example 1 of the present invention and the strength average hydrodynamic diameter (Dh) distribution (ac) and the corresponding line scan profile of Cy1, Cy2, and Cy3 micelles am.
5 is an actual image of the encapsulation efficiency of Nile Red (a) and the encapsulation efficiency of Cy1, Cy2, and Cy3 in Cy1, Cy2, and Cy3 of Example 1 of the present invention and E60, P37, and F47 of Comparative Examples 2 to 4 of the present invention. (b) are shown respectively.
Figure 6 is a schematic diagram (a) and chemical structure change (b) of drug release profiles based on pyrene fluorescence method using Cy1, Cy2, Cy3 prepared in Example 1 of the present invention and E60, P37, F47 of Comparative Examples 2 to 4 ) and drug release kinetics (c, d) of micelles using Cy1, Cy2, Cy3 prepared in Example 1 loaded with pyrene at pH 5.0 and pH 7.4 and E60, P37, F47 of Comparative Examples 2 to 4 It shows the result of the analysis.
7 shows the results of FRET-based encapsulation stability analysis in PBS and FBS using fluorescence emission spectra (λex = 450 nm) for Cy1, Cy2, and Cy3 prepared in Example 1 of the present invention.
8 shows the results of in vitro cell viability analysis determined by MTT assay using HeLa cells for Cy1, Cy2, and Cy3 prepared in Example 1 of the present invention.

Hereinafter, the present invention will be described in more detail as an embodiment.

The present invention relates to a pH-sensitive amphiphilic block copolymer, a method for preparing the same, and a polymeric micelle containing the pH-sensitive amphiphilic block copolymer.

The pH-sensitive amphiphilic block copolymer according to the present invention is prepared by copolymerizing polycyclohexyloxyethyl glycidyl ether (PCHGE) having hydrophobicity at one end of a hydrophilic polymer to prepare a pH-sensitive amphiphilic block copolymer. Self-assembling polymeric micelles that can be encapsulated can be formed by strong intermolecular hydrophobic interactions, and have excellent stability to the human body. In addition, the amphiphilic block copolymer has excellent loading efficiency due to CHGE having high hydrophobicity. Furthermore, it can be applied to the polyether-based bio field using this.

In addition, the polymeric micelles according to the present invention have excellent encapsulation stability and biocompatibility, and can effectively release drugs by controlling the decomposition rate of micelles by controlling the pH or the number of CHGE monomers due to the acetal bond that reacts to the pH of CHGE. can be adjusted so that For example, the polymeric micelles may be maintained at 37° C. for up to 14 days to enable sustained release and control of degradation kinetics. In addition, the excellent biocompatibility of the polymeric micelles has the advantage of being applicable as a drug delivery carrier.

The present invention provides a pH-sensitive amphiphilic block copolymer in which polycyclohexyloxyethyl glycidyl ether (PCHGE) having hydrophobicity is copolymerized by anionic ring-opening polymerization at one end of a hydrophilic polymer.

The hydrophilic polymer may be a polymer with excellent human body stability, and specific examples are poly(ethylene glycol) methyl ether, polyethylene glycol diacrylate, polyethylene glycol, polyethyleneimide, polyamino acid, polymethyl methacrylate, polyethylene oxide, poly It may be at least one selected from the group consisting of vinyl alcohol, polyacrylic acid, and polyvinylpyrrolidone. Preferably, it may be at least one selected from the group consisting of poly(ethylene glycol) methyl ether, polyethylene glycol diacrylate, polyethylene glycol, and polyethylene oxide, and most preferably poly(ethylene glycol) methyl ether.

The polycyclohexyloxyethyl glycidyl ether (PCHGE) is hydrophobic because it contains an epoxy monomer, and has a characteristic that can be encapsulated by a hydrophobic interaction between molecules. In addition, when forming polymeric micelles, various physiologically active ingredients such as pyrene-based organic substances, molecular imaging markers, contrast agents or therapeutic substances are chemically bound to the polycyclohexyloxyethyl glycidyl ether (PCHGE) to be located in the core of the polymeric micelles. can

The pH-sensitive amphiphilic block copolymer may be poly (ethylene glycol) methyl ether-block-polycyclohexyloxyethyl glycidyl ether (PEG-b-PCHGE) represented by Formula 1 below.

[Formula 1]

(In Formula 1, R ₁ is a cyclic group ( ), n and m are the repeating numbers of each repeating unit, n is an integer from 50 to 200, m is an integer from 3 to 46, and n: m is 1: 0.01 to 1: 0.9.)

Preferably, in Formula 1, R ₁ is a cyclic group ( ), n is an integer of 70 to 180, m is an integer of 3 to 46, and n:m may be 1:0.06 to 1:0.7. More preferably, in Formula 1, n is an integer of 90 to 160, m is an integer of 18 to 35, and n:m may be 1:0.1 to 1:0.5. Most preferably, in Formula 1, R ₁ is a cyclic group ( ), n is an integer of 110 to 120, m is an integer of 26 to 30, and n:m may be 1:0.2 to 1:0.3. In particular, when the number of repetitions of the polycyclohexyloxyethyl glycidyl ether (PCHGE) is less than 3, the number average molecular weight and glass transition temperature are significantly lowered, and the hydrophobic interaction is rapidly reduced, making it difficult to form polymeric micelles. If it exceeds 46, the amount of polymer used to form micelles may be reduced.

The pH-sensitive amphiphilic block copolymer has a number average molecular weight (Mn, _NMR ) of 6,000 to 20,000 g/mol, a molecular weight dispersion (Mw/Mn) of 1.08 to 1.19, and a glass transition temperature (Tg) of -40 to -25 °C. The number average molecular weight may be preferably 6,400 to 14,000 g/mol, more preferably 8,500 to 12,000 g/mol, and most preferably 9,800 to 11,000 g/mol. The molecular weight dispersion may be preferably 1.09 to 1.13, most preferably 1.1 to 1.12. In addition, the glass transition temperature (Tg) may be preferably -35.3 to -28.6 ℃, most preferably -31 to -29.5 ℃.

At this time, when the number average molecular weight, molecular weight dispersion and glass transition temperature of the pH-sensitive amphiphilic block copolymer satisfy all of the above physical property ranges, the average diameter of the polymeric micelles is uniformly formed to ensure uniform drug loading efficiency. , can maintain a constant decomposition rate.

The pH-sensitive amphiphilic block copolymer has a critical micelle concentration (CMC) of 1.4 to 13 mg/L, preferably 1.64 to 9.49 mg/L, more preferably 1.91 to 5.24 mg/L, and most preferably 2.13 mg/L. to 2.81 mg/L.

The pH-sensitive amphiphilic block copolymer forms self-assembled micelles when the pH is in the range of 7.2 to 7.6, and the micelles may be disintegrated when the pH is in the range of 1 to 5. The pH-sensitive amphiphilic block copolymer forms stable micelles in the pH range of 7.2 to 7.6, which is the pH range of normal cells in the body, and when the pH is less than 7.0 indicated by abnormal cells such as cancer cells, the micelle structure collapses, thereby chemically forming micelles. Due to the release of the diagnostic agent coupled to, cancer cells can be targetedly diagnosed and can serve as a carrier for drug release of a therapeutic agent for treatment accordingly. That is, in an acidic environment where the pH is in the range of 1 to 5, the acetal bond reacting to the pH of CHGE in the amphiphilic block copolymer is hydrolyzed and the micelles may be disintegrated while having a double hydrophilic group. That is, the pH-sensitive amphiphilic block copolymer can control the decomposition rate of the polymeric micelles by adjusting the pH.

On the other hand, the present invention is a pH-sensitive amphiphilic block copolymer; and a molecular imaging marker, a contrast agent, or a therapeutic agent bonded to polycyclohexyloxyethyl glycidyl ether (PCHGE) of the pH-sensitive amphiphilic block copolymer.

The polymeric micelles form micelles when injected into the body, and when they reach a place with a low pH locally, such as cancer cells, the micelles collapse, thereby providing molecular imaging markers for diagnosis of underlying diseases, contrast agents, or treatment of diseases. There is an advantage in that target-directed drug delivery is possible by releasing a therapeutic agent for the target.

The molecular imaging markers are for diagnosis of diseases, and include pyrene, pyrenebutyric acid, pyrene methylamine, 1-Aminopyrene, and pyrene-1-boronic acid. acid), iron oxide, and manganese oxide, and preferably pyrene.

The therapeutic substance is for treatment of disease and may be at least one selected from the group consisting of peptides, proteins, anticancer agents, anti-inflammatory agents, antibiotics, antibacterial agents, hormones, genes, and vaccines, but is not limited thereto.

The polymeric micelles may have an average diameter of 20 to 200 nm, preferably 26 to 119 nm, more preferably 60 to 105 nm, and most preferably 82 to 87 nm. At this time, if the average diameter of the polymeric micelles is less than 20 nm, it may be difficult to support a sufficient amount of drug inside the core, and if it exceeds 200 nm, the micelle form is excessively increased in CHGE, a hydrophobic core, compared to the hydrophilic polymer shell. may collapse

The polymeric micelles may be formulated for injection, transdermal or oral use so that they can be administered through oral, transdermal, ophthalmic, subcutaneous, intravenous, intramuscular or intraperitoneal routes, but are not limited thereto.

In addition, the present invention provides a pH-sensitive targeting delivery system comprising the polymeric micelles.

In addition, the present invention comprises the steps of synthesizing cyclohexyloxyethyl glycidyl ether (CHGE); and preparing a pH-sensitive amphiphilic block copolymer by adding a hydrophilic polymer, the cyclohexyloxyethyl glycidyl ether (CHGE), and a catalyst to an organic solvent and conducting anionic ring-opening polymerization. A method for producing a block copolymer is provided.

In the synthesizing of cyclohexyloxyethyl glycidyl ether (CHGE), glycidol and cyclohexylvinyl ether may be added to a dichloromethane solvent and polymerized.

In the step of preparing the amphiphilic block copolymer, the hydrophilic polymer and cyclohexyloxyethyl glycidyl ether (CHGE) are mixed at a molar ratio of 1:5 to 60, preferably at a molar ratio of 1:10 to 55, more preferably at a molar ratio of 1: 45 to 50 molar ratio, most preferably 1: 25 to 35 molar ratio may be mixed. In particular, if the cyclohexyloxyethyl glycidyl ether (CHGE) is less than 5 molar ratio, the hydrophobic interaction is too weak and micelle formation may be difficult. can

The catalyst may be at least one selected from the group consisting of t-BuP ₄ , t-BuP ₁ and EtP ₂ , and preferably t-BuP ₄ .

The step of preparing the amphiphilic block copolymer is polymerized at 50 to 100 ° C for 24 to 72 hours, preferably at 52 to 70 ° C for 30 to 68 hours, and most preferably at 55 to 65 ° C for 46 to 50 hours. can At this time, if both the polymerization temperature and polymerization time conditions are not satisfied, the cross-linking between the hydrophilic polymer and CHGE may not sufficiently occur, or the thermal and chemical stability of the amphiphilic block copolymer obtained may be reduced due to excessive cross-linking. .

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited by the following examples.

Example 1

(1) material

All reagents and solvents were purchased from Sigma-Aldrich, Alfa Aesar or Tokyo Chemical Industry and were used as is unless otherwise specified. Dry toluene and dichloromethane were dried and degassed using a solvent solvent system (Vacuum Atmospheres, USA), and immediately collected and used. CDCl ₃ and D ₂ O, deuterated NMR solvents, were purchased from Cambridge Isotope Laboratories, Inc. CHGE was used for polymerization after drying on molecular sieve 4A.

(2) Synthesis of cyclohexyloxyethyl glycidyl ether (CHGE) monomer

[Scheme 1]

A solution of glycidol (15.0 g, 0.202 mol) and cyclohexylvinyl ether (17.03 g, 0.135 mol) dissolved in dichloromethane (200 mL) was put into a round bottom flask and stirred at room temperature for 30 minutes to prepare a mixed solution. To the mixed solution, p-toluenesulfonic acid (0.23g, 1.35 mmol) was slowly added, and the mixture was stirred at 0 °C for 2 hours. Then, a saturated NaHCO ₃ solution was additionally added, and the mixed solution was stirred for 1 hour. Then, the aqueous phase of the mixed solution was extracted with dichloromethane, and the organic layer was extracted with deionized water.

The organic layer obtained was dried over Na ₂ SO ₄ . The dried organic phase was concentrated under reduced pressure, and the residue was purified by flash column chromatography using ethyl acetate/hexane (1:9 v/v) as an eluent to obtain 22.98 g (85%) of CHGE monomer as a colorless liquid. The CHGE monomer was then further purified by distillation. Successful synthesis of the CHGE monomer was confirmed by 1H and 13C NMR, correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and electrospray ionization mass spectrometry (ESI-MS) (see FIG. 2 below).

1H NMR (400 MHz, CDCl3): δ 4.86 (qd, J = 5.2, 0.6 Hz, 1H), 3.81 (dd, J = 11.3, 3.3 Hz, 1H), 3.69 (dd, J = 11.4, 3.4 Hz, 1H) ), 3.60 - 3.49 (m, 1H), 3.41 (dd, J = 11.3, 6.0 Hz, 1H), 3.18 - 3.09 (m, 1H), 2.83 - 2.76 (m, 1H), 2.64 (dd, J = 5.1 , 2.7 Hz, 1H), 2.60 (dd, J = 5.0, 2.7 Hz, 1H), 1.93 - 1.80 (m, 2H), 1.79 - 1.67 (m, 2H), 1.58 - 1.49 (m, 1H), 1.41 - 1.11 (m, 8H). 13C NMR (101 MHz, CDCl3): δ 98.14, 98.04, 74.49, 74.34, 65.19, 64.34, 50.94, 50.87, 44.73, 44.60, 33.44, 33.34, 32.65, 32.54, 25. 64, 24.39, 24.17, 20.81, 20.58. MS (m/z + Na+, ESI+) calcd for C11H20O3, 223.14; found, 223.13

(3) synthesis of mPEG-b-PCHGE block copolymer

[Scheme 2]

(In Scheme 3 above, n is 114 and m is 10 (Cy1), 30 (Cy2) or 50 (Cy3), respectively.)

In the synthesis of the block copolymer, poly(ethylene glycol) methyl ether (mPEG) was used as a macroinitiator. mPEG (1.0 g, 0.2 mmol) was placed in the flask under nitrogen flow. Toluene (3.0 mL) was then added to the flask and the mixture was stirred at 70 °C for 30 minutes. Then 0.25 mL of t-BuP ₄ in n-hexane (0.8 M, 0.2 mmol) was added to the solution. After 30 min, CHGE (2.0 g, 10.0 mmol) was added dropwise to the solution over 6 h using a syringe pump. After stirring at 60 °C for 2 days, polymerization was terminated by adding benzoic acid (0.02 g, 0.2 mmol). The reaction mixture was passed through an alumina pad with dichloromethane. The solutions were evaporated to dryness to obtain Cy3 (Table 1; 1.98 g) (yield: 71%). The number average molecular weight (Mn) of Cy3 was determined to be 14,000 g/mol as calculated from NMR data (FIG. 2) using the following equation. In addition, Cy2 was obtained in the same manner as above, except that mPEG (1.0 g, 0.2 mmol) and CHGE (1.2 g, 6.0 mmol) were mixed. In addition, Cy1 was obtained in the same manner as above, except that mPEG (1.0 g, 0.2 mmol) and CHGE (0.4 g, 2.0 mmol) were mixed.

Number of repeat units (CHGE) = 45 (integral value of methine peak, 45H, 4.80 ppm and methyl of mPEG, 3H, 3.38 ppm); Mn = (200.28 g/mol (molecular weight of CHGE monomer) x 45 (number of repeat units)) + 5000 g/mol (molecular weight of mPEG) = 14012 g/mol. Considering the error range of NMR integration, 14000 g/mol was used as the Mn value of Cy3.

1H NMR (400MHz, CDCl3) δ 4.85 - 4.74 (m, 45H), 3.96 - 3.40 (m, 734H), 3.39 (s, 3H), 1.86 (s, 94H), 1.73 (s, 92H), 1.61 - 1.47 (m, 44H), 1.42 - 1.09 (m, 385H).

Comparative Example 1

(3) PCHGE homopolymer synthesis

[Scheme 3]

(In Scheme 3 above, n is 25 or 50.)

A solution of 0.125 mL of t-BuP ₄ (0.80 M, 0.1 mmol) in n-hexane was added to a solution of benzyl alcohol (10.4 μL, 0.1 mmol) in toluene under a nitrogen atmosphere. CHGE (1.0 g, 5 mmol) was then added dropwise to the solution with a syringe pump, followed by polymerization over 6 hours. After stirring at room temperature for 24 hours, polymerization was stopped by adding benzoic acid (12.2 mg, 0.1 mmol). After quenching, the obtained polymer was then passed through an alumina pad using dichloromethane. Then, the polymer was evaporated to dryness to obtain PCHGE (500 mg) (yield: 50%). The number average molecular weight of PCHGE ₅₀ (Table 1) was 10100 g/mol as calculated from the NMR data shown in Figure 2 using the following equation.

Number of repeat units (CHGE) = 50 (integral value of methine peak, 50H, 4.78 ppm and methylene of benzyl alcohol, 2H, 4.53 ppm); Mn = (200.28 g/mol (molecular weight of CHGE monomer) ㅧ 50 (number of repeating units)) + 108.14 g/mol (molecular weight of benzyl alcohol) = 10122.14 g/mol. Considering the error range of NMR integration, the Mn value of PCHGE ₅₀ was 10100 g/mol. In addition, PCHGE ₂₅ was obtained in the same manner as above, except that CHGE (0.5 g, 2.5 mmol) was mixed.

1H NMR (400MHz, CDCl3): δ 4.79 (dd, J = 10.1, 6.4Hz, 50H), 4.54 (s, 2H), 3.78 - 3.34 (m, 312H), 1.84 (s, 105H), 1.72 (s, 109H), 1.52 (d, J = 7.1 Hz, 53H), 1.40 - 1.07 (m, 438H).

Comparative Examples 2 to 4

Blocks of commercially available mPEG ₁₁₄ -b-PCHGE ₆₀ (Comparative Example 2, E60), mPEG ₁₁₄ -b-PCHGE ₃₇ (Comparative Example 3, P37) and mPEG ₁₁₄ -b-PCHGE ₄₇ (Comparative Example 4, F47) Copolymers were prepared respectively.

Experimental Example 1: NMR spectrum analysis

1H NMR spectra of the CHGE monomer, the mPEG-b-PCHGE block copolymer, and the PCHGE homopolymer prepared in Example 1 and Comparative Example 1 were analyzed, and the results are shown in FIG. 1 .

1 is a 1H NMR spectrum analysis result of CHGE monomer (a), PCHGE homopolymer (b), and mPEG-b-PCHGE block copolymer (c) prepared in Example 1 and Comparative Example 1. Referring to FIG. 1, in the case of the PCHGE homopolymer (b) of Comparative Example 1, the disappearance of epoxide peaks a and b of the monomers at 2.60, 2.80 and 3.15 ppm indicated that the monomers were converted into polymers. Also benzyl protons (x; 4.52 to 4.55 ppm), polyether backbone protons (a, b and c; 3.40 to 3.75 ppm), methine protons (d; 4.75 to 4.85 ppm), and methyl and cyclohexyloxy protons (eg : g, h and i, 1.10 to 1.90 ppm), it was confirmed that other peaks of the PCHGE homopolymer appeared clearly. The theoretical number average molecular weight (Mn, NMR) was calculated as the integral ratio of benzylic protons and methine protons in the 1H NMR spectrum.

Also, referring to (c) of FIG. 1, the 1H NMR spectrum of the mPEG-b-PCHGE block copolymer clearly showed each characteristic peak according to the structure: methyl proton (x; 3.38 ppm) of the mPEG macroinitiator. , methine protons (d; 4.75 to 4.80 ppm), polyether backbone protons (y, z; 3.40 to 3.75 ppm), methyl and cyclohexyloxy protons (eg g, h and i; 1.10 to 1.90 ppm). The degree of polymerization (DP) of the amphiphilic block copolymer was determined by the ratio of the methine peak of CHGE (g; 4.75 to 4.80 ppm) and the methine peak of mPEG (3.38 ppm). Although not specified in Experimental Example 1, the GPC results of the mPEG-b-PCHGE block copolymer showed a single mode distribution with narrow dispersion (D <1.13) in all items.

Experimental Example 2: Analysis of Mn, Tg and CMC physical properties

Number average molecular weight (Mn), glass transition temperature (Tg) and critical micelle concentration (CMC) for the CHGE monomer, mPEG-b-PCHGE block copolymer, and PCHGE homopolymer prepared in Example 1 and Comparative Examples 1 to 4, respectively. ) was measured. The results are shown in Table 1.

[measurement method]

Number average molecular weight (Mn) was measured by b1H NMR spectroscopy, cGPC (THF, RI signal, PMMA standard) and dGPC (DMF, RI signal, PMMA standard).

The glass transition temperature (Tg) was measured at a rate of 10 °C/min by differential scanning calorimetry (DSC). Critical micelle concentration (CMC) values were calculated by fluorescence spectroscopy using pyrene as a probe.

Since the critical micelle concentration (CMC) is the minimum micelle-forming polymer concentration for micelle formation and is an important parameter for predicting micelle stability, the CMC value of the amphiphilic block copolymer was measured using pyrene as a fluorescent probe. The specific photoluminescence of pyrene was plotted for various concentrations of each polymer as the ratio of I339/I332 (intensity of 339 at 332 nm) when the pyrenes were close to each other, such as the hydrophobic core of the self-assembled polymeric micelles. For the specific critical micelle concentration (MC) measurement method using the pyrene fluorescence method, polymer solutions in dimethylformamide (DMF) at various concentrations (0.001 mg/L to 1000 mg/L) were prepared. 10 μl of pyrene solution (5.2 mg/L in DMF) was added to the mPEG-b-PCHGE block copolymer solution and the mixture was stirred at room temperature for 30 minutes. A total of 5 mL of deionized water was then added to the solution at a rate of 0.5 mL/min using a syringe pump. The solution was allowed to equilibrate overnight. Fluorescence of each pyrene-containing polymeric micelle solution (different concentrations) was measured at an emission wavelength of 372 nm using a fluorometer in a 1 × 1 cm quartz cell. The following parameters were selected. Emission wavelength = 372 nm, excitation wavelength range = 300 - 360 nm, data interval = 0.5 nm. The ratio of fluorescence intensity at wavelengths of 339 and 332 nm was plotted against the polymer concentration and the CMC was determined at the inflection point.

According to the results of Table 1, the polymerization conversion rate of each composition was 99% or more. In the case of Example 1, it was confirmed that the number average molecular weight (Mn, _NMR ) increased as the number of CHGEs increased. In addition, the CMC values of Example 1 were 9.49 ± 2.61 (Cy1), 2.47 ± 0.34 (Cy2), and 1.64 ± 0.18 mg/L (Cy3), respectively. That is, the CMC value of the amphiphilic block copolymer decreased as the number of hydrophobic blocks increased. In particular, in the case of Cy2, it was found that the loading efficiency of the critical micelle concentration compared to the number of CHGE monomers was the best. In addition, in the case of Comparative Examples 2 to 4, compared to Cy2 and Cy3 of Example 1, the number average molecular weight (Mn, _NMR ) was relatively low, and in the CMC value, the E60 of EEGE and TPGE (10.3 mg / L ), P37 (9.79 mg/L) and F47 (113.6 mg/L), and it was confirmed that they were higher than Cy2 and Cy3 of Example 1. Through this, in the case of Example 1, it was found that the CMC value was relatively much lower than that of Comparative Examples 2 to 4, indicating that the hydrophobicity of the CHGE monomer was high.

Meanwhile, the glass transition temperature (Tg) of the PCHGE homopolymer of Comparative Example 1 increased from -36.8 °C to -31.5 °C as the polymerization degree of CHGE increased. From these results, it was found that the higher the chair-to-chair stacking of cyclohexyl groups in CHGE, the higher the Tg value. Similarly, in the amphiphilic block copolymer of Example 1, as the degree of polymerization of CHGE increased, the Tg value of the polymer increased from -35 °C (Cy1) to -28 °C (Cy3). In addition, it was confirmed that the Tg of the mPEG-b-PCHGE was similar to that of the TFGE block copolymer but lower than that of the TPGE block copolymer. It was found that although the Tg was increased due to the cyclohexyl group of CHGE, the result was lower than the Tg of TPGE because CHGE had a more linear structure than TPGE.

Experimental Example 3: MALDI-ToF spectroscopy and hydrolysis reaction analysis

After confirming the synthesis of the PCHGE homopolymer of Comparative Example 1 through NMR and GPC in Experimental Examples 1 and 2, MALDI-ToF spectroscopy was performed to confirm whether CHGE was cross-linked to the PCHGE homopolymer. The results are shown in Figure 2.

2 shows the results of MALDI-ToF mass spectrum analysis of the mPEG-b-PCHGE block copolymer prepared in Example 1 from 2750 to 4500 Da. In FIG. 2 , each symbol corresponds to an intact polymer (red circle) and a partially decomposed polymer fragment (blue triangle and green square) during measurement. Referring to FIG. 2, in the MALDI-ToF spectrum, the molecular weight of 3734.9 g/mol is benzyl alcohol initiator (108.06 g mol-1), 18 monomers (200.14 g mol-1) and Na ⁺ as a counter ion. Corresponding to the polymer, two types of small peaks were observed due to partial decomposition of cyclohexyloxy groups during the measurement. In particular, it was found that the polymerization was successful because the interval between the major signal and the minor signal corresponded to the molecular weight of the functional CHGE monomer.

Before investigating the self-assembly behavior of the amphiphilic block copolymer prepared in Example 1, the hydrolysis dynamics of the CHGE monomer was investigated through quantum mechanical calculation using density functional theory, and the results are shown in FIG. 3. Here, the hydrolysis kinetics were compared with other types of monomers with acetal groups, such as ethoxyethyl propylether (EEPE), tetrahydropyranyl propylether (TFPE) and tetrahydropyranyl propylether (TPPE).

Figure 3 schematically shows the energy diagram (a) of the hydrolysis reaction coordinates of various model acetal side chain compounds (CHPE, TPPE, TFPE, EEPE) and the proposed hydrolysis mechanism (b) of CHPE. 3, green molecules are water molecules involved in the hydrolysis of CHPE. In addition, cyclohexyloxy propyl ether (CHPE) was synthesized as a new model compound in consideration of the side chain structure formed after polymerization of the CHGE monomer.

Referring to FIG. 3, in the first step, 25 explicit water molecules (25 H ₂ O) and additional protons were included to calculate the proton energy barrier. Optimized structures of model compounds with the lowest energy structures were obtained, including transition state structures before and after protonation. In particular, protonation of the outer shell of the water molecule for CHPE revealed the high hydrophobicity of the model compound. Then, the reaction energy barrier (ΔE) was calculated with limited optimization according to the fixed distance between oxygen and proton in the CHPE structure. The reactive energy profile showed the protonation of the model acetal compound along the OH bond length. In addition, the CHPE (29.0 kcal / mol) has the highest energy barrier (ΔE) for protonation than all other monomers including the TPPE (24.2 kcal / mol), TFPE (11.2 kcal / mol) and EEPE (10.0 kcal / mol) ) was shown. This means that CHPE has the highest hydrophobicity than other acetal model compounds.

In addition, the hydrophobicity of each monomer was evaluated using the partition coefficient (logP) with the calculation model ALOGPS 2.1. The calculated logP values were 2.93 (CHPE), 1.72 (TPPE) and 1.31 (TFPE) ²⁴ , supporting the trend observed in the reactive energy profile.

In general, total hydrolysis of an acetal bond involves two steps: (1) protonation and (2) bond cleavage. The rate-limiting step of total hydrolysis was observed to be the protonation reaction with the highest value of 29.0 kcal/mol for CHPE. Then hydrolysis of the protonated CHPE compound (i.e., H+-CHPE; FIG. 4B) using two explicit water molecules for hydrolysis of the protonated CHPE compound through an activation energy barrier of about 1.0 kcal/mol. Reaction energy barriers and transition states were calculated in the step. This process was similarly observed for ring opening of H + -TFPE and H + -TPPE with a single transition state involving significantly lower energy barriers (0.37 kcal/mol and 0.83 kcal/mol, respectively) compared to the protonation step. In addition, no activation energy barrier was observed for C-O bond breakage for H+ -EEPE, indicating that simultaneous hydrolysis was followed by a protonation step.

Motivated by computational analysis of the model compound, hydrolysis of the CHPE model compound was performed by in situ 1 H NMR spectroscopy under acidic conditions in sodium acetate-d3/acetic acid-d4 buffer at pH 5.0. However, the limited solubility of CHPE monomers in aqueous buffer prevented hydrolysis monitoring unlike other model acetal compounds. Together with the calculation results, the hydrolysis of the acetal group in CHPE was prolonged due to the high hydrophobicity of the CHPE model compound.

Experimental Example 4: DLS and AFM evaluation

For the Cy1, Cy2 and Cy3 micelles prepared in Example 1, the size and shape of the micelles were confirmed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results are shown in FIG. 4 .

4 is an AFM image (d-f) of Cy1, Cy2, and Cy3 micelles prepared in Example 1, and the intensity average hydrodynamic diameter (Dh) distribution (a-c) of Cy1, Cy2, and Cy3 micelles with corresponding line scan profiles. Referring to (a-c) of FIG. 4, the concentrations of all polymers performed in the DLS measurement were above the CMC value (1 mg/mL). In addition, the hydrodynamic diameter (Dh) of each micelle measured by DLS was 26.72 ± 1.99 (Cy1), 85.06 ± 0.54 (Cy2), and 118.56 ± 4.21 nm (Cy3), and the diameter increased as the number of CHGE increased. Through this, it was inferred that the size of the micelles increased due to the presence of steric hindrance in the hydrophobic core of the micelles due to the increase in bulky hydrophobic units (CHGE).

In addition, referring to (d-f) of FIG. 4, in the AFM image, DAFM = 55.16 ± 1.8 nm for Cy1, DAFM = 91.27 ± 2.3 nm for Cy2, and DAFM = 130.89 ± 1.4 nm for Cy3, respectively, the spherical shape and constant diameter of the micelles The distribution was shown, and it was confirmed that it showed a level similar to the DLS result.

Experimental Example 5: Evaluation of encapsulation efficiency

Cy1, Cy2 and Cy3 micelles prepared in Example 1 and E60, P37, and F47 of Comparative Examples 2 to 4 were evaluated for encapsulation efficiency of the micelles, and the results are shown in FIG. 5.

The encapsulation efficiency (EE) of the micelles was measured as follows. 0.1 mL of a solution of the model hydrophobic dye Nile Red (50 μg/mL in acetone) was added to a solution of mPEG-b-PCHGE block copolymer (5 mg) in acetone (0.1 mL) and the mixture was stirred at room temperature for 30 minutes. . A total of 5 mL deionized water was added to the solution at a rate of 0.5 mL/min using a syringe pump. The solution was then allowed to equilibrate overnight and the acetone evaporated with the cap open. After filtration using a 0.45 μm syringe filter to remove unloaded Nile Red, the solution was lyophilized and redissolved in 1.0 mL acetone. The amount of Nile Red encapsulated in the micelles was measured using a fluorometer in a 1 x 1 cm quartz cell, and the encapsulation efficiency (EE) was calculated by Equation 1 below.

[Calculation 1]

5 is an actual image of the encapsulation efficiency of Nile Red (a) and the encapsulation efficiency of Cy1, Cy2, and Cy3 in Cy1, Cy2, and Cy3 of Example 1 and E60, P37, and F47 of Comparative Examples 2 to 4 (b) ) are shown, respectively. Referring to FIG. 5, the loading efficiencies of Nile Red in Cy1, Cy2, and Cy3 of Example 1 were 20.35% (Cy1), 57.59% (Cy2), and 61.47% (Cy3) in each micelle core. In other studies ( Biomacromolecules 2014, 15, (11), 4281-4292.), the encapsulation efficiency was from a minimum of 26% to a maximum of 53.5%, confirming that it was lower than the CHGE-based micelles of Example 1. In addition, compared to previous studies, E60, P37, and F47 of Comparative Examples 2 to 4 using an acetal-containing polymer were 10.9% (E60), 16.6% (P37), and 9.8% (F47), and the CHGE-based micelles had a hydrophobic interaction of the core. It was found that the effect was increased. In addition, the loading efficiency of the micelles increased as the CHGE monomer unit increased, and was saturated at Cy2, which was in good agreement with the CMC value. Through this, it was confirmed that introducing a highly hydrophobic and bulky CHGE monomer into the polymer could form micelles with excellent loading efficiency.

Experimental Example 6: Evaluation of decomposition rate in an acidic environment

Polymeric micelles are promising as drug delivery vehicles, but the stability of micelles is still a challenge for biological applications. In particular, considering the various biological substances that can be encountered in circulation, the encapsulation of hydrophobic drugs is affected by various proteins in the blood. To this end, Cy1, Cy2, Cy3 prepared in Example 1 and E60, P37, and F47 of Comparative Examples 2 to 4 were applied to drug release profiles based on pyrene fluorescence method in various environments (FBS, PBS, pH 5.0 and pH 7.4). The degradation rate of the micelles was evaluated. The results are shown in FIG. 6 .

6 is a schematic diagram of drug release profiles based on pyrene fluorescence method using Cy1, Cy2, and Cy3 prepared in Example 1 and E60, P37, and F47 of Comparative Examples 2 to 4 (a) and chemical structural changes (b) Analysis of drug release kinetics (c, d) of micelles using Cy1, Cy2, Cy3 prepared in Example 1 loaded with pyrene at pH 5.0 and pH 7.4 and E60, P37, and F47 of Comparative Examples 2 to 4 that showed the result. Referring to (a, b) of FIG. 6, the amphiphilic block copolymer of Example 1 exists in the form of micelles, and then swells as the acetal bond is protonated by water molecules in the solvent. When a swelling phenomenon occurs and hydrolysis in the form of polyglycerol (PG) occurs, the micelles are decomposed and the drug is released.

(c, d) of FIG. 6 shows the results of monitoring the stability of each pyrene-containing micelle for the change in I339/I332 fluorescence ratio intensity in acidic (pH 5.0) and normal (pH 7.4) environments at a temperature of 37 °C. As a result of investigating the micelle degradation in the normal (pH 7.4) environment, the fluorescence ratio intensity of I339 / I332 in the Cy1 to Cy3 micelles of Example 1 did not change, which means that the micelles were stable in the pH 7.4 environment. On the other hand, in the micelles in the acidic (pH 5.0) environment, the CHGE portion was changed to hydrophilic linear PG by hydrolysis, and decomposition was observed due to disruption of the hydrophobic/hydrophilic balance. As the micelles responded to low pH, pyrene encapsulated in the core was released, Cy1 was most significantly reduced, and Cy2 and Cy3 also decreased in strength over time. In addition, in the case of E60, P37, and F47 of Comparative Examples 2 to 4, it was confirmed that the pyrene strength quickly disappeared over time due to the rapid decomposition of micelles in an acidic (pH 5.0) environment.

In particular, the release rate of pyrene was slower when the micelles contained more units of CHGE monomer. For example, Cy1 degraded the fastest and released most of the pyrene in 4 days, Cy2 completely degraded after 12 days and the longest after 14 days. These results are consistent with the CMC values of Cy1 to Cy3, indicating that the release kinetics can be controlled. In addition, in the case of Cy3, which has the largest hydrophobic site, it has the strongest hydrophobic interaction in the core, showing the longest release time due to high-density packing, and it can be seen that long-term sustained release is possible.

Experimental Example 7: Evaluation of encapsulation stability in a biological environment (PBS and FBS)

The stability of the micelles of Cy1, Cy2, and Cy3 prepared in Example 1 was investigated in various biological environments (PBS and FBS). A fluorescence resonance energy transfer (FRET)-based method was used to confirm the stability of the micelles in a biological environment. The stability of the micelles was analyzed by loading FRET pairs of DiO (FRET-donor) and DiI (FRET-acceptor) into the hydrophobic core of the micelles. For reference, if the micelles are stable under harsh conditions, efficient energy transfer from DiO (FRET-donor) to DiI (FRET-acceptor) occurs because the FRET pair is located close to the core and strong emission occurs near 560 nm. However, if the micelles are unstable, they become FRET pairs. Only strong DiO emission (FRET donor) at 510 nm appears as the position is free and no energy is transferred between the pairs. The analysis results are shown in FIG. 7 .

7 shows the results of FRET-based encapsulation stability analysis in PBS and FBS using fluorescence emission spectra (λex = 450 nm) for Cy1, Cy2, and Cy3 prepared in Example 1. Referring to FIG. 7, Cy1, Cy2, and Cy3 micelles showed strong emission only at about 550 nm in phosphate-buffer saline (PBS) for 192 hours, and no peak was observed at 510 nm. As described above, since DiO and DiI are close to the core of the micelles, it can be seen that these results result from efficient energy transfer, and it means that the micelles have excellent stability in PBS.

In addition, almost no peak change was observed in Cy3 micelles during the entire measurement time, but a greater peak change was observed in Cy2 and Cy1 micelles after 120 hours. Through this, it was found that the more the number of hydrophobic units, the denser the DiO and DiI of the core, the higher the stability of the micellar core and no peak variation was observed.

In addition, as a result of measuring the experiment up to 72 hours due to the formation of aggregates of proteins and lipids present in fetal bovine serum (FBS), strong emission was observed at 550 nm from all micelles for 72 hours, and strong emission at 510 nm No peak change was observed. Similar to the PBS data, the peak change was only observed for Cy1 but not for Cy2 and Cy3. These results indicated that all micelles were unaffected by serum proteins and could safely hold the payload in the core. In addition, overall micelles showed excellent stability in a biological environment, and it was confirmed that they deteriorated in response to low pH.

Experimental Example 8: In vivo cytotoxicity evaluation

In order to evaluate the applicability of Cy1, Cy2, and Cy3 prepared in Example 1 in a drug delivery system, a cell viability test of micelles was performed. In particular, polymeric micelles should have high biocompatibility for drug delivery systems. In order to measure the cell viability of the micelles synthesized as a drug delivery system, the MTT assay for Hela cells was investigated.

8 shows the results of in vitro cell viability analysis determined by MTT assay using HeLa cells for Cy1, Cy2, and Cy3 prepared in Example 1 above. Referring to FIG. 8, it was confirmed that, in the case of Cy1, Cy2, and Cy3, the cell viability was all maintained at about 100% even when the micelle solution was treated at various concentrations or exposed to a concentration of 1000 μg/mL. These results indicate that each of the amphiphilic block copolymers of Cy1, Cy2, and Cy3 is suitable for application as a drug delivery carrier. That is, it was found that the Cy1, Cy2, and Cy3 exhibited excellent cell viability to Hela cells, making them suitable as drug delivery systems.

Claims (15)

A pH-sensitive amphiphilic block copolymer in which polycyclohexyloxyethyl glycidyl ether (PCHGE) having hydrophobicity is copolymerized by anionic ring-opening polymerization at one end of a hydrophilic polymer,
The pH-sensitive amphiphilic block copolymer is poly (ethylene glycol) methyl ether-block-polycyclohexyloxyethyl glycidyl ether (PEG-b-PCHGE) represented by Formula 1 below,
The pH-sensitive amphiphilic block copolymer has a critical micelle concentration (CMC) of 1.64 to 9.49 mg / L pH-sensitive amphiphilic block copolymer.
[Formula 1]

(In Formula 1, R1 is a cyclic group ( ), n and m are the repeating numbers of each repeating unit, n is an integer from 70 to 180, m is an integer from 3 to 46, and n: m is 1: 0.06 to 1: 0.7.)
delete delete According to claim 1,
The pH-sensitive amphiphilic block copolymer has a number average molecular weight (Mn, _NMR ) of 6,000 to 20,000 g/mol, a molecular weight dispersion (Mw/Mn) of 1.08 to 1.19, and a glass transition temperature (Tg) of -40 to -25 ° C. pH-sensitive amphiphilic block copolymer.
delete According to claim 1,
The pH-sensitive amphiphilic block copolymer forms self-assembling micelles when the pH is in the range of 7.2 to 7.6, and the micelles are disintegrated when the pH is in the range of 1 to 5. .
The pH-sensitive amphiphilic block copolymer according to any one of claims 1, 4 and 6; and
Polymer micelles containing; a molecular imaging marker, a contrast agent, or a therapeutic agent bonded to polycyclohexyloxyethyl glycidyl ether (PCHGE) of the pH-sensitive amphiphilic block copolymer.
According to claim 7,
The molecular image markers include pyrene, pyrenebutyric acid, pyrene methylamine, 1-Aminopyrene, pyrene-1-boronic acid, iron oxide and oxide A polymeric micelle that is at least one selected from the group consisting of manganese.
According to claim 7,
The therapeutic substance is at least one selected from the group consisting of peptides, proteins, anticancer agents, anti-inflammatory drugs, antibiotics, antibacterial agents, hormones, genes, and vaccines.
According to claim 7,
The polymeric micelles are polymeric micelles having an average diameter of 20 to 200 nm.
According to claim 7,
The polymeric micelles are formulated for injection, transdermal or oral use so that they can be administered through oral, transdermal, ocular, subcutaneous, intravenous, intramuscular or intraperitoneal routes.
A pH-sensitive targeting delivery system comprising the polymeric micelles according to claim 7 .
synthesizing cyclohexyloxyethyl glycidyl ether (CHGE); and
Preparing a pH-sensitive amphiphilic block copolymer by adding a hydrophilic polymer, the cyclohexyloxyethyl glycidyl ether (CHGE), and a catalyst to an organic solvent, and performing anionic ring-opening polymerization,
The pH-sensitive amphiphilic block copolymer is poly (ethylene glycol) methyl ether-block-polycyclohexyloxyethyl glycidyl ether (PEG-b-PCHGE) represented by Formula 1 below,
The pH-sensitive amphiphilic block copolymer has a critical micelle concentration (CMC) of 1.64 to 9.49 mg / L, a method for producing a pH-sensitive amphiphilic block copolymer.
[Formula 1]

(In Formula 1, R ₁ is a cyclic group ( ), n and m are the repeating numbers of each repeating unit, n is an integer from 70 to 180, m is an integer from 3 to 46, and n: m is 1: 0.06 to 1: 0.7.)
According to claim 13,
In the step of preparing the pH-sensitive amphiphilic block copolymer, the hydrophilic polymer and cyclohexyloxyethyl glycidyl ether (CHGE) are mixed in a molar ratio of 1: 5 to 60 of the pH-sensitive amphiphilic block copolymer. manufacturing method.
According to claim 13,
The step of preparing the pH-sensitive amphiphilic block copolymer is a method for producing a pH-sensitive amphiphilic block copolymer of polymerizing at 50 to 100 ° C. for 10 to 30 hours.