Protein-encapsulating polymer micelles

Abstract

To provide a micelle capable of increasing retentivity in blood and efficiently releasing a protein in an acidic condition for increasing a therapeutic effect by a therapeutic protein.SOLUTION: A polymer composite containing a protein and a block copolymer represented by the following formula (1) is provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention utilizes block copolymers to provide a stable, in vivo solution. The present invention relates to a protein-encapsulating polymeric micelle that can improve the quality of the protein. The disclosure is incorporated herein by reference in its entirety. [Background technology]

[0002] Proteins are physiologically active substances that exist everywhere in the body, and they are used to treat cancer and autoimmune diseases. It is used to treat various intractable diseases such as autoimmune diseases and metabolic disorders. Systemic administration of proteins alone is subject to enzymatic degradation and renal excretion, and may also be immunogenic. Therefore, the development of a delivery vehicle is necessary for the biological application of proteins. Proteins that have been introduced with the biocompatible polymer poly(ethylene glycol) (PEG) The development of protein-PEG conjugates is underway, and their interaction with proteolytic enzymes and immune cells is being investigated. Protein challenges due to reduced activity and increased size [1-4] It is possible to overcome In fact, many protein-PEG conjugates have been approved by the FDA, and Multi-billion dollar pharmaceutical market [5,6] However, PEGylation of proteins can Elementary degradation, renal excretion, and immunogenicity [7,8] While suppressing the irreversible chemical modification of proteins Protein inactivation by decoration and protein function [6,9] insufficient spatiotemporal control of Therefore, we have developed a method to convert proteins into materials through reversible chemical bonds. This suppresses protein expression in normal tissues while targeting the target tissue.

[10] specifically released Development of delivery vehicles that can

[0003] Stimulus-responsive nanocarriers target tissues [4,11] By sensing physiologically active substances in This allows the protein to be released specifically to the target tissue while retaining its activity. In such nanocarriers, the block copolymers and proteins form autonomous associations. Core-shell polymeric micelles incorporate environmentally responsive moieties into the core-forming chains of block copolymers By doing so, external stimuli [4] It can induce the release of proteins in response to high molecular weight. One example of an external stimulus that the micelles can respond to is pH, which is a factor in many diseases (e.g., cancer). or autoimmune diseases) are normal tissue (pH 7.4) [12,13] It has a lower pH (pH 6.5-7.2) than

[0004] On the other hand, the present inventors have previously reported on pH-responsive maleic anhydride derivatives. [14-16] by the amino group By adding PEG-polycation to the protein whose carboxyl group has been converted, poly We demonstrated that ionic complex (PIC) type polymeric micelles can be prepared. The core stably encapsulates proteins at the pH of normal tissue (pH 7.4), but at the acidic pH of target tissue (pH H 6.5-7.2) releases the protein by cleavage of the pH-responsive maleic anhydride derivative I succeeded in doing so. However, for medical applications, it is necessary to improve blood retention and increase accumulation in target tissues. is important. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, in order to increase the therapeutic effect of therapeutic proteins, it is necessary to increase blood retention and It is important to develop micelles that allow efficient release of proteins in acidic conditions. [Means for solving the problem]

[0006] The present invention introduces a pH-responsive maleic anhydride derivative into the core-forming chain of a block copolymer, Increased micelle stability by forming reversible covalent bonds with amino groups of proteins The aim was to achieve efficient release of proteins under acidic conditions. Further micelle formation is achieved by PIC formation between the amino groups of the core-forming chains and the carboxyl groups of the protein. The micelle structure was stabilized by covalent bonding and PIC formation, and blood retention was increased. The aim is to make it big.

[0007] That is, the present invention is as follows. [1] A polymer complex comprising a protein and a block copolymer represented by the following formula (1): [ka] [In the formula, R 1 and R 2 are each independently a hydrogen atom or an optionally substituted carbon atom A linear or branched alkyl group having 1 to 12 carbon atoms, or an azide, amine, maleimide, ligase, represents a band or labeling agent, R 3 represents a compound represented by the following formula (I): [ka] (In the formula, R a and R b are each independently a hydrogen atom or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group , a heterocyclic alkyl group, a hydroxy group, an alkoxy group, or an aryloxy group. Ta, R a and R b are bonded to each other and form an aromatic ring or a cycloalkyl ring together with the carbon atoms to which they are bonded. An alkyl ring may be formed. a and R b The bond between the carbon atoms to which , may be a single bond or a double bond.) L 1 is NH, CO, or the following formula (11): -(CH2) p1 -NH- (11) (In the formula, p1 represents an integer of 1 to 6.) or a group represented by the following formula (12): -L 2a -(CH2) q1 -L 3a - (12) (In the formula, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a NH or represents CO. q1 represents an integer of 1 to 6. represents a group represented by m1 and m2 each independently represent an integer of 0 to 500 (provided that the sum of m1 and m2 is 10 to 500). m3, m4 and m5 each independently represent an integer of 1 to 5, and n represents an integer of 0 to 500. Represents an integer. The symbol " / " indicates that the (m1 + m2) monomer units on either side of it can be arranged in any order. It means that.] [2] The compound represented by formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig): The complex according to [1], wherein the complex is at least one type. [ka] [3] The compound represented by formula (I) is a compound represented by the following formula (Ia) or (Ib): [2] The complex according to claim 1. [ka] [4] The block copolymer represented by formula 1 is represented by the following formula (2), A complex of [ka] [5] The method according to [1], wherein a protein is covalently bound to a block copolymer represented by formula 1. A complex of [6] The complex according to [5], wherein the covalent bond is cleaved in a pH-dependent manner. [7] A method for detecting a cell surface or intracellular component comprising the polymer conjugate according to any one of [1] to [6]. and a protein delivery device selected from the outside of a cell. [8] A block copolymer represented by the following formula (1) is used to bind to a cell surface, an intracellular or extracellular A protein delivery kit for any of the following: [ka] [In the formula, R 1 and R 2 are each independently a hydrogen atom or an optionally substituted carbon atom A linear or branched alkyl group having 1 to 12 carbon atoms, or an azide, amine, maleimide, ligase, represents a band or labeling agent, R 3 represents a compound represented by the following formula (I): [ka] (In the formula, R a and R b are each independently a hydrogen atom or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group , a heterocyclic alkyl group, a hydroxy group, an alkoxy group, or an aryloxy group. Ta, R a and R b are bonded to each other and form an aromatic ring or a cycloalkyl ring together with the carbon atoms to which they are bonded. An alkyl ring may be formed. a and R b The bond between the carbon atoms to which , may be a single bond or a double bond.) L 1 is NH, CO, or the following formula (11): -(CH2) p1 -NH- (11) (In the formula, p1 represents an integer of 1 to 6.) or a group represented by the following formula (12): -L 2a -(CH2) q1 -L 3a - (12) (In the formula, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a NH or represents CO. q1 represents an integer of 1 to 6. represents a group represented by m1 and m2 each independently represent an integer of 0 to 500 (provided that the sum of m1 and m2 is 10 to 500). represents an integer; m3, m4, and m5 each independently represent an integer of 1 to 5; and n represents an integer of 0 to 500. Represents. The symbol " / " indicates that the (m1 + m2) monomer units on either side of it can be arranged in any order. It means that.] [9] The compound represented by formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig): The kit according to [8], wherein the kit contains at least one type of compound. [ka]

[10] The compound represented by formula (I) is a compound represented by the following formula (Ia) or (Ib): [9 ] A kit described in. [ka]

[11] The block copolymer represented by formula 1 is represented by the following formula (2), The kit described. [ka] [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows a pH-responsive protein-encapsulating micelle formed by polyion complex formation and pH-responsive amide bond. [Figure 2] Self-assembly of PEG-p(Lys-CDM) in buffer solutions of different pHs. a) Count rates normalized by the count rate of PEG-p(Lys-CDM) at pH 7.4. PEG-p(Lys-CDM) was added to 10 mM acetate buffer (pH 4 or 5) containing 150 mM NaCl or 10 mM phosphate buffer (pH 6.5 or 7.4) containing 150 mM NaCl at a concentration of 1 mg / mL, vortexed for 1 minute, and then incubated for 1 hour. DLS measurements were performed. Data are shown as mean ± standard deviation (n = 3). b) Size distribution of empty-PIC micelles formed at pH 7.4. [Figure 3]This figure shows the stability of empty micelles prepared in a pH 7.4 buffer solution. Empty micelles were added to 10 mM phosphate buffer (pH 6.5 (gray dots) or 7.4 (black dots)) containing 150 mM NaCl to a final concentration of 0.5 mg / mL, and DLS measurements were performed. a) Particle size, b) PDI, and c) count rate normalized by the count rate before dilution. [Figure 4] In vitro cytotoxicity of PEG-p(Lys-CDM) (gray line) against HEK 293 cells after 48 hours of incubation with different polymer concentrations. PEG-p(Lys) (black line) was used as a control. Data are presented as mean ± standard deviation (n=4). [Figure 5] Figure 1 shows the stability of protein-encapsulated micelles in solutions of different pH. Particle size (a) and PDI (b) of myo / m (gray and black circles) and CC-myo / m (open circles) in 10 mM phosphate buffer at pH 6.5 (gray line) and pH 7.4 (black line). [Figure 6] Figure 1 shows the stability of myo / m after dilution into 10 mM phosphate buffers containing 600 mM NaCl at different pH levels. Particle size (a) and normalized count rate (b) of myo / m in pH 6.5 (gray line) and pH 7.4 buffers (black line) demonstrate the collapse of myo / m in pH 6.5 buffer. [Figure 7] Figure 1 shows the release of Alexa Fluor 647-labeled myoglobin from myo / m (pH 7.4, pH 6.5) in 10 mM phosphate buffer containing 150 mM NaCl. [Figure 8]Figure 1 shows the evaluation of myoglobin activity. a) UV / Vis absorption spectra of oxygenated myoglobin after O2 gas introduction (gray line) and reduced myoglobin after Ar gas introduction (black line). Inset: Spectrum of myoglobin released from micelles from 500 to 600 nm. b) UV / Vis absorption spectra of native oxygenated myoglobin after O2 gas introduction (gray line) and reduced myoglobin after Ar gas introduction (black line). Inset: Spectrum of native myoglobin from 500 to 600 nm. cd) Absorbance at 414 nm of released myoglobin (c, white mark) and native myoglobin (d, black mark) upon alternating introduction of O2 (square mark) and Ar (triangle mark) gases. [Figure 9] Figure 1 shows the blood retention of fluorescently labeled myoglobin, CC-myo / m, and myo / m, measured by IV-CLSM. a) Myoglobin alone, b) CC-myo / m, and c) myo / m prepared using Alexa Fluor 647-labeled myoglobin (red). d)-e) d) CC-myo / m and e) myo / m prepared using Alexa Fluor 647-labeled polymer (red). Microscopic images (a-e, left panels) immediately after sample administration were normalized and quantified using the fluorescence intensity in the vein (red trapezoid) and skin (green trapezoid) (a-e, right panels). [Figure 10] Microdistribution of fluorescently labeled myoglobin, CC-myo / m, and myo / m in the kidney, liver, and spleen. a)-c) Myoglobin alone, b) CC-myo / m, and c) myo / m prepared using Alexa Fluor 647-labeled myoglobin (red). d)-e) Alexa Fluor 647-labeled polymer (red). d) CC-myo / m and e) myo / m prepared using Alexa Fluor 647-labeled polymer (red). Cell nuclei were stained with Hoechst (cyan). Scale bar: 100 μm. [Figure 11]Chemical analysis of PEG-p(Lys-TFA). a) H-NMR spectrum of PEG-p(Lys-TFA) in DMSO-d. b) GPC chromatogram of PEG-p(Lys-TFA), showing a monomodal peak and narrow molecular weight distribution (Mw / Mn = 1.03). (Flow rate: 0.8 mL / min, Mobile phase: DMF solution containing 10 mM LiCl) [Figure 12] Figure 1 shows chemical analysis of PEG-p(Lys). a) H-NMR spectrum of PEG-p(Lys) in DO. b) GPC chromatogram of PEG-p(Lys). (Flow rate: 0.75 mL / min, mobile phase: acetate-buffered saline (pH 3.3) with 10 mM acetate and 500 mM NaCl.) [Figure 13] Figure 1 shows the characterization of PEG-p(Lys-CDM). a) H-NMR spectrum of PEG-p(Lys-CDM) in DMSO-d6, b) aqueous-phase GPC chromatogram of PEG-p(Lys-CDM). (Flow rate: 0.75 mL / min, eluent: acetate-buffered saline (pH 3.3) with 10 mM acetate and 500 mM NaCl.) [Figure 14] Characterization of PEG-p(Lys-CDM). a) H-NMR spectrum of PEG-p(Lys-CDM) in 10 mM deuterated phosphate buffer (0.70 ml) at 25 °C, pH 7.4. The peak intensity of the polyamino acid protons was lower than expected due to the limited mobility of the protons in the polymer caused by micelle formation. b) H-NMR spectrum of PEG-p(Lys-CDM) after incubation with 2 M deuterated hydrochloric acid (volume ratio 1:35) for 10 minutes. The peak intensity of the polyamino acid protons recovered by approximately 75% after acid treatment, suggesting that the collapse of micelles under acidic conditions increased the mobility of the protons in the polymer. [Figure 15] FIG. 1 shows the size distribution of 1 mg / mL PEG-p(Lys-CDM) in DMEM. [Figure 16]TEM images of lysozyme (left), myoglobin (center), and BSA-encapsulated micelles (right). Scale bar: 50 nm. The micelle morphology was observed by TEM (JEM-1400, JEOL). Protein-encapsulated micelles were stained with phosphotungstic acid (PTA) (2%, w / v) and placed on a 400-mesh copper grid. Images were taken at 50,000x magnification. [Figure 17] FIG. 1 shows the size distribution of IL-12-encapsulated micelles. [Figure 18] FIG. 1 shows the release of IL-12 from IL-12-encapsulating micelles. [Figure 19] FIG. 1 shows the amount of INF-γ secreted in mouse splenocytes by IL-12-encapsulating micelles. DETAILED DESCRIPTION OF THE INVENTION

[0009] Therapeutic proteins hold promise for the treatment of intractable diseases, but their systemic administration There are various challenges with the use of riboflavin, such as instability, short half-life, and non-specific immune responses. Therefore, the approach of delivering proteins via stimuli-responsive nanocarriers is expected to be effective in targeting tissues. This may be an effective strategy for tissue-selectively enhancing the activity of proteins in the body. In this study, proteins and blockers were used to release the loaded proteins in a pH-dependent manner. A polyion complex is formed between the polymer and the copolymer, and the complex is cleavable under a specific pH condition. We have developed polymeric micelles that have the ability to encapsulate proteins via photoinitiated bonding.

[0010] The carboxydimethylmaleic anhydride (CDM)-amide bond is It is stable but is cleaved at pH 6.5, the pathophysiological pH of tumors and inflamed tissues. Therefore, CDM was selected as the pH-responsive functional group. In the present invention, a 45% CDM-added polymer was used. By using poly(ethylene glycol)-poly(L-lysine) block copolymer, It was encapsulated into different proteins with various molecular weights and isoelectric points with an efficiency of over 50%. Using robin-encapsulated micelles (myo / m) as a model, we investigated the effects of robin on the physiologic conditions of micelles. The stability of the cells, as well as the collapse of the micelles and release of functional myoglobin at pH 6.5, were confirmed. Furthermore, myo / m is a molecule formed by electrostatic interactions only, without covalent bonds, and myoglobin alone. Therefore, the blood half-life was improved compared to micelles assembled with other drugs. The usefulness of this system for in vivo delivery of therapeutic proteins was demonstrated.

[0011] CDM-amide bond at pH 6.5 [17-19] are unstable at pathological pH, and therefore conjugated amino acids In the present invention, CDM was selected as the pH-responsive moiety to enable the release of the compound. Therefore, the resulting protein-encapsulating micelles form a stable cross-linked core at physiological pH. However, at pH 6.5, it decomposes into free block copolymers and active proteins (Figure 1). In this study, we evaluated the ability of these micelles to encapsulate various proteins. The inventors have used micelles encapsulating myoglobin and IL-12 as models to In vitro stability and protein release at different pH levels, and in vivo after systemic administration The blood retention of

[0012] 1. Polymer composite of the present invention The polymer complex of the present invention is a protein-encapsulating polymeric micelle complex (polyion complex). It is a specific cationic polymer (block copolymer (PIC)) (also called polymers, graft copolymers, etc.) and proteins (more on proteins later). Includes:

[0013] (1) Cationic polymer The specific cationic polymer that is a component of the PIC of the present invention is at least partially composed of a polycation. The cationic polymer is, for example, a polyethylene glycol monoacrylate. A block copolymer or graft having a ethylene glycol (PEG) moiety and a polycation moiety. The polymer may be any suitable polymer depending on the application of the PIC of the present invention. A new mode can be selected.

[0014] The structures (for example, the degree of polymerization) of the above-mentioned PEG and polycation are not limited, and any Among them, polycations having cationic groups on the side chains can be selected. The term "cationic group" used herein means a polypeptide in which a hydrogen ion is Not only groups that are already cationic through coordination, but also groups that become cationic when a hydrogen ion is coordinated Such cationic groups include all known groups. The polypeptide having a thionic group in the side chain is a polypeptide having a known amino acid (lysine) having a basic side chain. In addition to those made up of peptide bonds between amino acids such as arginine, histidine, etc., various amino acids are The peptide bond has a cationic side chain (e.g., the side chain of aspartic acid or glutamic acid). Also included are those substituted to have the following structure:

[0015] Specific examples of the specific cationic polymer include those represented by the following general formula (1): A preferred example is a block copolymer. [ka]

[0016] In the structural formula of general formula (1), the block portion having the number of repeating units (degree of polymerization) n is a PEG portion. The number of repeating units is the block portion (general formula (1)) that combines the portion m1 and the portion m2. In the figure, the part shown in [ ] is the polycation part. The symbol " / " in the formula indicates that the order of the monomer units shown on either side of it is arbitrary. For example, a block portion composed of monomer units A and B is represented by [- When written as [(A)a- / -(B)b-], it means a number of A's and b number of B's, totaling (a+b). may be linked randomly in any order. (However, all As and Bs are linked in a linear chain.)

[0017] In general formula (1), R 1 and R 2 are each independently a hydrogen atom or an optionally substituted a linear or branched alkyl group having 1 to 12 carbon atoms, or an azide, an amine, or a maleimide; Represents a functional group such as a ligand or labeling agent. Examples of the linear or branched alkyl group having 1 to 12 carbon atoms include a methyl group, Ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group , n-pentyl group, n-hexyl group, decyl group, and undecyl group. Examples of the substituent of the alkyl group include an acetalized formyl group, a cyano group, a formyl group, Carboxyl group, amino group, alkoxycarbonyl group having 1 to 6 carbon atoms, alkoxycarbonyl group having 2 to 7 carbon atoms Silamido group, siloxy group, silylamino group, and trialkylsiloxy group (each alkyl The siloxy groups each independently have 1 to 6 carbon atoms.

[0018] A ligand molecule refers to a compound used to target a specific biomolecule, e.g. For example, antibodies, aptamers, proteins, amino acids, small molecules, and biopolymer monomers. Examples of the labeling agent include rare earth fluorescent labeling agents, coumarin, dimethylamino Sulfonylbenzoxadiazole (DBD), dansyl, nitrobenzoxadiazole (NB D), pyrene, fluorescein, fluorescent proteins, etc. However, it is not limited to the above.

[0019] When the substituent is an acetal-protected formyl group, this substituent is preferably an acetal-protected formyl group. Another substituent, a formyl group (or aldehyde group; -CHO), when hydrolyzed under certain conditions ) can be converted to the above substituents (especially R 1 the substituent in Or in the case of a carboxyl group or an amino group, for example, an antibody or or fragments thereof, or proteins with other functions or targeting properties. It is possible.

[0020] In general formula (1), R 3 represents a compound represented by the following general formula (I). [ka] In the above formula (I), R a and R b are each independently a hydrogen atom or an optionally substituted alkyl groups, alkenyl groups, cycloalkyl groups, aryl groups, aralkyl groups, acyl groups , a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group, or an aryloxy group Also, R a and R b and bond together with the carbon atoms to which they are bonded to form an aromatic ring or In addition, in formula (I), R a and R b are combined with each other The bond between the carbon atoms may be a single bond or a double bond, but is not limited thereto. In formula (I), in order to show both bonding modes, the carbon atoms are connected by a single bond. It is represented by a solid line and another dashed line.

[0021] L 1 is represented by NH, CO, or the following general formula (11): -(CH2) p1 -NH- (11) (In the formula, p1 represents an integer of 1 to 6.) or a group represented by the following general formula (12): -L 2a -(CH2) q1 -L 3a - (12) (In the formula, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a NH or represents CO. q1 represents an integer of 1 to 6. represents a group represented by the following formula:

[0022] In the above formula (1), m1 and m2 each independently represent an integer of 0 to 500 (provided that m1 and m2 are The sum of m3, m4 and m5 represents an integer of 1 to 5. In the above formula (1), n ​​represents the number of repeating units (degree of polymerization) of the PEG moiety, and more specifically, represents an integer of 1 to 500 (preferably 100 to 400, more preferably 200 to 300).

[0023] The molecular weight (Mn) of the cationic polymer represented by the general formula (1) is not limited, but is preferably 23,000. Preferably, the molecular weight of each block is 28,000 to 45,000, and more preferably 28,000 to 34,000. Regarding the locking portion, the molecular weight (Mw) of the PEG portion is preferably 8,000 to 15,000. , more preferably 10,000 to 12,000, and the molecular weight (Mn) of the polycation moiety is 15 or less overall. It is preferably 1,000 to 30,000, and more preferably 18,000 to 22,000.

[0024] The method for producing the cationic polymer represented by general formula (1) is not limited, but may be, for example, a method for producing a cationic polymer represented by general formula (1) 1 A segment (PEG segment) containing the PEG chain block portion is synthesized in advance, and One end of the PEG segment (R 1 The desired monomers are polymerized in order at the opposite ends of the polymer chain. A method of substituting or converting the side chain to contain a cationic group as needed, or A segment and a block portion having a side chain containing a cationic group are synthesized in advance, The methods and conditions for various reactions in the production method include: can be appropriately selected or set in consideration of common practices.

[0025] In one embodiment of the present invention, the compound represented by formula (I) is represented by the following formulas (Ia) to (Ig): At least one of the compounds. [ka]

[0026] In a preferred embodiment of the present invention, the compound of formula (I) is represented by the following formula (Ia) or (Ib): It is a compound that can be [ka]

[0027] In formula (I), the substituents are saturated or unsaturated acyclic or cyclic hydrocarbon groups. In the case of a hydrocarbon group, it may be either linear or branched. For example, C1-C 20 Alkyl groups, C2-C 20 Alkenyl groups, C4-C 20 Cycloalkyl groups, C6-C 18 Aryl group, C6-C 20 Aralkyl groups, C1-C 20 Alkoxy groups, C6-C 18 An example is an aryloxy group.

[0028] The compound represented by formula (I) is used as a charge control agent. The compound represented by formula (I) is a salt It converts the overall charge of a basic or neutral protein into the charge of an acidic protein. In other words, the charge control agent of the present invention is a charge control agent that controls the total charge to be positive (+) or neutral. The protein in the charged state is then charged so that the total charge is on the negative (-) side. It can be said that the total charge is converted by controlling the amount of charge. Specifically, the compound represented by the formula (I) or a derivative thereof is an amino acid sequence of an amino acid sequence contained in a protein. It binds to amino groups (positively charged groups) and makes the entire protein negatively charged. For this purpose, the bond can be formed by, for example, reacting the compound of formula (I) with The amino acid bonded to the amino group in the protein (covalent bond) forms the following formula (I'): This is done by taking structure. [ka]

[0029] Regarding the above bond, for example, when the compound represented by the formula (I) is a compound represented by the formula (Ib) and (Ic), In the case where the compound is a compound represented by the formula (I'), the structure represented by the formula (I') after the bonding is: It is as follows. [ka]

[0030] In a further embodiment of the present invention, the block copolymer of formula 1 is represented by formula 2: can be. [ka]

[0031] (2) Protein In the PIC of the present invention, the protein constituting the core portion is a protein represented by the above-mentioned formula (I). The protein whose overall charge has been converted by the compound shown in ), and specifically, the total charge is the total charge (positive side) of a basic or neutral protein. or neutral state) to become negative, similar to the total charge of the acidic protein. Any protein that has been converted so that the total charge is negative is acceptable. Proteins as a whole can be said to be anionic substances (polyanions). Therefore, due to electrostatic interaction with the polycation portion in the cationic polymer, Easily forms micellar complexes, which are difficult to form with basic or neutral proteins can be done.

[0032] The types of proteins used in the present invention include those that are originally included in basic or neutral proteins. The protein used in the present invention is not limited to a simple protein. In addition, glycoproteins and lipid proteins are also included. The protein is not limited to a full-length amino acid sequence, but also includes partial fragments and peptides thereof. Furthermore, proteins consisting of two or more molecules (dimers), and their partial sequences or full-length sequences are also The proteins used in the present invention also include fusion proteins of natural amino acids. The present invention is not limited to those composed of unnatural amino acids at least in part as constituents. Furthermore, the protein used in the present invention may, if necessary, be modified with: The present invention also includes proteins to which various labeling substances have been added as appropriate. Examples of such proteins include hemoproteins, various cytokines, various enzymes, and antibodies (e.g., nuclear Examples of antibodies include, but are not limited to, antibodies against membrane pore complexes or antibody fragments. It's not that.

[0033] (3) Polyion Complex (PIC) The PIC of the present invention comprises a protein and a portion of the cationic polymer (polycation portion) described above. The core part is formed by electrostatic interaction with the other part ( The core-shell structure is such that the core (including the PEG moiety) forms a shell around the core. These can be called micelle-type complexes.

[0034] The PIC of the present invention can be used, for example, to dissolve a protein and a cationic polymer in an arbitrary buffer (e.g., It can be easily prepared by mixing in a cationic surfactant (e.g., Tris buffer). The mixing ratio of the polymer and the protein is not limited. In the present invention, for example, The total number of cationic groups (e.g., amino groups) in the copolymer (N) and the number of cationic groups in the protein The ratio of the total number of carboxyl groups (C) to the total number of carboxyl groups (N / C ratio) can be 0.1 to 200, and can be 0.5 to 100. When the N / C ratio is in the above range, the free cationic This is preferable in that the polymer can be reduced. The carboxyl groups in the corresponding proteins form ionic bonds through electrostatic interactions. means a group which can

[0035] The size of the PIC of the present invention can be measured, for example, by dynamic light scattering (DLS) measurement, but is not limited thereto. The particle size is preferably 5 to 200 nm, more preferably 10 to 100 nm.

[0036] The PIC of the present invention releases the encapsulated protein after being introduced into cells. When this happens, the pH environment in the cytoplasm changes (to a weakly acidic environment (e.g., pH 5.5)). The compound represented by formula (I) dissociates from the protein (the bond is broken). As a result, the overall charge of the protein (total charge) is The total charge of the protein is restored to its original value. can be present in a state in which its structure, activity, etc. are regenerated.

[0037] 2. Protein Delivery Devices In the present invention, a protein delivery system including the above-mentioned polyion complex (PIC) is used. The protein delivery device of the present invention is a device for controlling the redox environment inside and outside the cell. By utilizing this change, the desired protein (charge conversion protein) encapsulated in the core part of the PIC can be A means for efficiently introducing the compound into any one of the cell surface, the inside of the cell, and the outside of the cell of the target cell. It can be used as such.

[0038] Specifically, a solution containing PICs encapsulating a desired protein is administered to a test animal, and the PICs are then administered to the test animal. The PIC then reaches the endosome. When this occurs, the compound represented by formula (I) is released from the protein, and the charge balance within the PIC changes. When the PIC collapses, proteins are released from the PIC, At the same time, the polymer dissociated from the PIC damages the endosomal membrane. Due to disruption of the endosomes, delivery of the released protein into the cytoplasm is achieved. For example, in the case of micelles containing cytokines such as IL-12, the protein is released outside the cell. The protein binds to receptors on the cell surface, making the cell surface the target of delivery. When delivering an enzyme that expresses its function inside a cell using a micelle, the protein The enzyme then releases the agonist, allowing the antibody to be delivered to the inside of the cell. When delivering a drug, it may target proteins secreted outside the cell, so it is important to Of course, two or three of the cell surface, intracellular and extracellular regions can be targeted. It is also possible to deliver a combination of the above.

[0039] The protein delivery device of the present invention can be used in humans, mice, rats, rabbits, pigs, dogs, and The method of administration to the subject animal is not limited to, and can be applied to various mammals such as cats. Usually, parenteral administration such as intravenous drip infusion is adopted, and various specifications such as dosage, frequency of administration and administration period are required. The conditions can be set appropriately depending on the type and condition of the subject animal.

[0040] The protein delivery device of the present invention delivers a desired protein to cells that cause various diseases. It can be used for treatments (e.g., enzyme replacement therapy, immunotherapy using antibodies, etc.) Therefore, the present invention provides a pharmaceutical composition containing the above-mentioned PIC (for example, for enzyme replacement therapy or immunotherapy). and methods for treating various diseases using the aforementioned PIC (e.g., enzyme It is also possible to provide treatments such as replacement therapy and immunotherapy using antibodies. The conditions are the same as above.

[0041] The pharmaceutical composition may contain excipients, fillers, bulking agents, binders, etc. that are commonly used in pharmaceutical manufacturing. Mixtures, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, antiseptics Preservatives, flavoring agents, soothing agents, stabilizers, isotonic agents, etc. are appropriately selected and used, and the mixture is prepared in the usual manner. The pharmaceutical composition can be prepared in the form of an intravenous injection (including drip infusion). ) may be employed and may be provided, for example, in unit dose ampoules or multi-dose containers.

[0042] 3. Protein Delivery Kit The protein delivery kit of the present invention is characterized by comprising the block copolymer. The kit is suitable for use in a desired method such as enzyme replacement therapy or an immunotherapy method using an antibody. The protein can be preferably used in various therapeutic methods.

[0043] In the kit of the present invention, the storage conditions of the cationic polymer are not limited, and the stability The form of the compound can be selected from a solution, a powder, or the like, taking into consideration storage stability and ease of use. The kit may contain other components in addition to the block copolymer. The components include, for example, various buffers, various proteins to be introduced into cells (charge conversion Examples of such materials include the protein, dissolution buffer, and instructions for use (instruction manual). The kit of the present invention is a polymer having a core portion of a desired protein to be introduced into target cells. It is used to prepare ionic complexes (PICs), which are then used to deliver PICs to target cells. It can be effectively used as a protein delivery device.

[0044] Example The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. It is not something that can be done.

[0045] 1. Materials and Methods 1.1. Materials α-Methoxy-ω-amino-poly(ethylene glycol) (MeO-PEG-NH2; Mn = 12,000) is a N-trifluoroacetyl-L-lysine N-carboxyanhydride was purchased from Company F (Tokyo, Japan). (Lys(TFA)-NCA) was purchased from Chuo Kasei Co., Ltd. (Tokyo, Japan). Oxalyl chloride, 2- Propion-3-methylmaleic anhydride, dichloromethane (CH2Cl2), N,N-dimethylformamide DMF, toluene, methanol, and deuterium oxide (99.8 atom % D) were purchased from Tokyo Chemical Industry Co., Ltd. Alexa Fluor 647 NHS ester (Succinimidyl Ester) was purchased from Sigma-Aldrich Co., Ltd. (Tokyo, Japan). was purchased from Thermo Fisher (Waltham, MA, USA), and modified with DMSO-d6 and Dulbecco's DMEM medium was purchased from Sigma Aldrich (St. Louis, MO, USA) and contained fetal bovine serum (FBS). was purchased from Sumitomo Dainippon Pharma (Osaka, Japan). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories. Dialysis membranes were purchased from Spectrum Laboratories Inc. (Rancho Domingo, CA). The filter was purchased from Vivaspin 6 centrifugal filter units (10,000 MWCO (molecular weight) Cut-off, 30,000 MWCO and 100,000 MWCO were purchased from Sartorius (Gottingen, Germany). I entered.

[0046] 1.2. Instruments Proton nuclear magnetic resonance ( 1 H-NMR spectra were obtained on a JEOL ECS-400 spectrometer with a frequency of 400 MHz. (JOEL Ltd., Japan), and chemical shifts were calculated as parts per million (ppm). The molecular weight distribution of the polymer was measured by gel permeation chromatography (GPC), and the organic phase GPC TSK Gel G4000H HR and G3000H HR A TOSOH HLC-8220 system (Tosoh, Japan) equipped with a column Poly(ethylene glycol) standards were used for calibration (Polymer Laboratories Aqueous phase GPC measurements were performed using a JASCO LC-EXTREMA system (JASCO, Japan) with size exclusion chromatography. A JASCO LC-EXTREMA system equipped with a Superdex 200-10 / 300GL column (GE Healthcare; USA) was used. The size distribution and zeta potential were measured by dynamic light scattering (DLS) and laser scattering, respectively. Measurements were performed via Doppler electrophoresis using a Zetasizer Nano-ZS (Malvern, UK). Fluorescence intensity from the fluorescamine assay was measured using an ND-3300 Nanodrop Fluorescence Spectrometer (Thermo Fisheye) UV / Vis spectroscopy was performed using a V-500 spectrophotometer (JASCO, Japan). Ta.

[0047] 1.3. Synthesis of PEG-poly(L-Lysine-CDM) block copolymer PEG-poly(L-lysine) block copolymer (PEG-p(Lys)) has been reported

[20] A slight modification to the method The following variations were made: MeO-PEG-NH2 (Mn = 12,000) was reacted with Lys(TFA)-NCA to form PEG-p(Lys -TFA) followed by deprotection of the trifluoroacetyl group. G-NH2 (1 g, 0.083 mmol) and Lys(TFA)-NCA (1.005 g, 3.75 mmol) were dissolved in 1 M thiourea solution containing DMF. After dissolving the NCA in water separately, the NCA solution was transferred to the PEG solution under an argon atmosphere and stirred at 35 °C for 3 days. The polymer was obtained as a white solid by precipitation in diethyl ether and drying under vacuum. It was recovered as a powder. The degree of polymerization was 1 The molecular weight distribution was determined by H-NMR (DMSO-d6, 80 °C). C (Mobile phase: 10 mM LiCl containing DMF; Temperature: 40 °C; Flow rate: 0.8 mL / min; Detector: Furthermore, the protecting group TFA was removed by treatment with 1 M NaOH in methanol at 35 °C overnight, followed by The ATP was removed by dialysis against water using a 6-8 kD MWCO dialysis membrane. The final product was obtained as a white powder. The components of the deprotected polymer were: 1 H-NMR (DO, 25 °C) So, we analyzed it.1 In the H-NMR spectrum, PEG-OC H 2C H 2 and -C3 of lysine H 6 Pro The composition of the PEG-p(Lys) block copolymer was determined by the intensity ratio of the peaks derived from PEG. The molecular weight distribution was analyzed by GPC (mobile phase: 10 mM acetate and 500 mM NaCl acetate buffer solution). Water (pH 3.3); room temperature; flow rate: 0.75 mL / min; detector: UV, wavelength 220 nm).

[0048] PEG-p(Lys-CDM) was prepared by reacting the acyl chloride of CDM with PEG-p(Lys). First, the acyl chloride of CDM (CDM-Cl) was prepared as previously reported.

[21] The preparation was based on the description in (2) with some modifications. -Propion-3-methylmaleic anhydride (CDM, 200 mg, 1.09 mmol) was dissolved in anhydrous toluene. The CDM was dissolved in anhydrous CH2Cl2 (15 mL) and then evaporated under vacuum. CDM (4 mL, 5.9 g, 46 mmol) was added and reacted with CDM at room temperature for 12 hours. Residual oxalyl chloride was removed by evaporation to give a clear oil. 2Cl2 (4 ml) was added to dissolve CDM-Cl, while CHCl2 (20 ml) was used to dissolve PEG-p(Lys) (200 ml) g, 0.011 mmol) was dissolved in the PEG-p(Lys) solution. The PEG-p(Lys) solution was then transferred to the CDM-Cl solution, and the reaction was allowed to stand at room temperature. After 12 hours, the product was recovered by diethyl ether precipitation and drying in vacuo overnight. The final product was 1 It was analyzed by H-NMR and GPC.

[0049] S1. Chemical reaction scheme, polymer synthesis and chemical analysis [ka]

[0050] [ka]

[0051] [ka]

[0052] 1.4. Core-crosslinked polyion complex (PIC) micelles without protein encapsulation (empty PIC micelles) Preparation of PEG-100 and its stability under various pH conditions The polymer solution (1 mg / mL) was dissolved in acetate buffer at pH 4 or 5, or phosphate buffer at pH 6.5 or 7.4. The polymers were prepared in a buffer solution (10 mM acetic acid or phosphoric acid with 150 mM NaCl). The solution was dissolved in buffer (vortex for 1 minute, incubate for 1 hour). The solution was then injected into a 0.22 μm syringe. The polymer solution was filtered through a filter, followed by DLS measurement. The samples were prepared in phosphate buffer (10 mM) at pH 7.4, before and after the addition of dihydrochloric acid (DCl). 1 H-NMR Analyzed.

[0053] Furthermore, the empty PIC micelles self-assembled in a pH 7.4 buffer solution were prepared at a final polymer concentration of 0.5 mg / ml of 10 mM phosphate buffer with 150 mM NaCl at pH 6.5 or 7.4. The stability of empty PIC micelles under these conditions was evaluated over time by DLS. Size distribution by intensity , polydispersity index (PDI), and count rate were evaluated.

[0054] 1.5. In vitro cytotoxicity The in vitro cytotoxicity of PEG-p(Lys-CDM) was evaluated against the human embryonic kidney 293 (HEK 293) cell line. PEG-p(Lys) was used as a control in this experiment. Cells were cultured in DMEM with 10% FBS. 3000 cells were seeded per well on a 96-well plate in medium and incubated at 37°C in 5% CO2 for 24 hours. The cells were then incubated with various concentrations of polymer. After incubation, cytotoxicity was assessed by measuring the formazan absorbance at 450 nm using CCK-8. Furthermore, PEG-p(Lys-CDM) block copolymer was dissolved in DMEM. The resulting solution was evaluated by DLS.

[0055] 1.6. Preparation and physicochemical evaluation of myoglobin-encapsulated micelles (myo / m) PEG-p(Lys-CDM) polymer (3 mg / mL) was dissolved in a pH 5 buffer (10 mM acetate) to form empty PIC micelles. To suppress the formation, a 0.1 molar equivalent myoglobin solution was prepared in buffer (10 mM phosphate, pH 8). After mixing the two solutions, the solution was adjusted to pH 7.4 and stirred for 6 hours. Phosphate buffered saline (10 mM phosphate containing 150 mM NaCl) was used to separate the pellets in a 100,000 MWCO centrifuge flask. The micelles were purified by ultrafiltration using a filter to remove unbound proteins. To evaluate the encapsulation efficiency, myoglobin was analyzed by Alexa Fl The mixture was labeled with uor 647 succinimidyl ester and subjected to GPC (150 ml of a pH 7.4 gel as the mobile phase). Analysis was performed using 10 mM phosphate buffer containing 10 mM NaCl; flow rate: 0.75 mL / min; room temperature.

[0056] The fluorescence detection settings were an excitation wavelength of 650 nm and an emission wavelength of 668 nm. The amount of protein absorbed was calculated by dividing it by the amount of protein added. The amount of Alexa Fluor 647-labeled myoglobin encapsulated per micelle was determined using a spectrophotometric method (FCS). The FCS experiment was performed using an MF-20 (Olympus, Japan) equipped with a laser beam with a wavelength of 633 nm. Furthermore, lysozyme and albumin were also analyzed by the same method. The micelles were encapsulated in a micelle, and the size of the micelles was determined by DLS.

[0057] 1.7. Preparation and physicochemical evaluation of CDM-modified myoglobin-encapsulating micelles (CC-myo / m) CDM-modified myoglobin (CC-myo)-encapsulated micelles (CC-myo / m) have been reported. [14,16] Just in the way Control micelles were prepared according to the same method with minor modifications. was dissolved in 0.1 M NaHCO3 buffer to prepare a 2 mg / mL solution, which was then stirred at 4°C for 30 minutes. Then, 50 molar equivalents of CDM was slowly added to the solution and stirred at 4°C for 2 hours. The bottled solution was purified by ultrafiltration using a 10,000 MWCO centrifugal filter. The efficiency of the fluorophores was measured using a Nanodrop fluorescence spectrometer (Thermo Fisher, USA). The percentage of converted amine was determined by the Lescamine method and the percentage of converted amine was determined by the previously published method.

[16] Calculated according to Subsequently, CC-myo / m was prepared by mixing PEG-p(Lys) with charge-converted myoglobin. and titrated into phosphate buffered saline at pH 7.4 with an N / C (amino group / carboxyl group) ratio of 2:1. Furthermore, PEG-p(Lys) and native myoglobin mixtures were mixed at the same polymer-to-protein molar ratio. The size distribution, polydispersity index (PDI) and zeta potential of micelles were measured using Zetas Analysis was performed using a HPLC Nano ZS.

[0058] 1.8. Stability of myoglobin-encapsulated micelles in buffers with different salt concentrations and pH values To test the in vitro stability of myo / m and CC-myo / m under different pH conditions, samples were The solution was diluted to a polymer concentration of 0.5 mg / mL. Micelles were incubated in 10 mM phosphate buffer at H 7.4 and measured by DLS over time. (25 °C). Size distribution, PDI and resulting count rates were recorded on a Zetasizer Nano ZS. Furthermore, the stability of micelles was investigated by shielding electrostatic interactions using a high-concentration salt buffer. Myo / m and CC-myo / m were prepared and diluted to a polymer concentration of 0.5 mg / mL. The solution was diluted with 20,000 MW HCl in 5 L of 10 mM phosphate buffer containing 600 mM NaCl at pH 7.4 and pH 6.5. The samples were taken from inside the dialysis cassette at different time points. The collapse of the micelles was followed by DLS analysis.

[0059] 1.9. Myoglobin release from myo / m under different pH conditions For Alexa Fluor 647-labeled myo / mM, use a dialysis cassette with a MWCO of 20,000 Da and a 5 L capacity. The sample was dialyzed in 10 mM phosphate buffer and 150 mM NaCl, pH 7.4 and pH 6.5, at room temperature. At predetermined time points, samples were taken from inside the dialysis cassette and analyzed using a NanoDrop 3300 fluorescence spectrometer. The fluorescence intensity was evaluated using

[0060] 1.10. Assessment of myoglobin activity Myoglobin was incubated overnight in 10 mM phosphate buffer + 150 mM NaCl at pH 6.5. The micelles were isolated by centrifugation followed by ultrafiltration through a 30,000 MWCO centrifugal filter. The filter permeate was collected and then passed through a 10,000 MWCO centrifugal filter. The myoglobin was concentrated to 0.05 mg / mL by ultrafiltration using a filtration method.

[22] to Spectroscopic measurements were performed with a UV / Vis spectrometer using a quartz cuvette with an optical length of 1 cm. The released myoglobin (0.05 mg / mL) was dissolved in 5 equivalents of sodium dithionite (NaSO4 The reduced myoglobin was then reduced by adding an aqueous solution of O2 to the mixture. The mixture was oxygenated by introducing argon for 2 minutes and then bubbling argon for 2 hours. The previously reported protocol

[22] The oxygenation / reduction cycle is repeated many times according to the As a control, native myoglobin at the same concentration was used.

[0061] 1.11. In vivo blood retention and biodistribution Alexa Fluor 647-labeled myoglobin was used to measure myo / m, CC-myo / m, and free myoglobin. and used a Nikon A1R intravital confocal laser scanning microscope (IV-CLSM) (Nikon, Japan). The blood retention and biodistribution of myoglobin were followed. 100 μL of sample solution containing 00 μg / mL of fluorescently labeled myoglobin was administered via the tail vein of anesthetized mice. Inject under anesthesia and observe capillaries in the earlobe

[23] The fluorescence intensity in the earlobe vein and skin was Twelve hours after injection, the mice were euthanized and organs (kidneys, liver, and spleen) were collected. The tissue was then excised and imaged ex vivo by IV-CLSM 30 min before euthanasia and organ harvest. Then, 100 μL of Hoechst 33342 solution was administered via the tail vein for nuclear staining. Using Alexa Fluor 647-labeled polymer and unlabeled myoglobin, we investigated the activity of micelles in blood. Myo / m and CC-myo / m were prepared to monitor the blood retention of the polymer. 100 μL of sample solution containing 1 mL of fluorescently labeled polymer was administered via the tail vein, and the earlobe capillaries were then irradiated. All animal experiments in this study were conducted in accordance with the University of Tokyo's regulations for handling experimental animals. This was carried out in accordance with the

[0062] 1.12. Protein and polymer labeling Protein labeling with Alexa Fluor 647 succinimidyl ester was performed according to the manufacturer's instructions. Briefly, 5 mg / ml protein was added to 0.15 M sodium bicarbonate. 0.5 molar equivalents of Alexa Fluor 647 succinimidyl ester was dissolved in DMSO buffer. F to prepare a 10 mg / ml solution. The above two solutions were mixed and reacted at room temperature for 1 hour. The solution was then applied to a Sephadex G-25 column and purified by gel permeation chromatography. After purification, the Alexa Fluor 647-labeled protein was lyophilized for further use. PEG-p(Lys) labeling and purification were carried out in the same manner as for proteins, except for PEG-p(Lys-CDM). Because of the self-assembly properties of , the labeling was carried out in 10 mM phosphate buffer (pH 6.5) and the free dye was After gel filtration to remove the polymer, the polymer solution was treated with 0.1 N HCl for 5 min and immediately frozen. It was dried.

[0063] 1.13. Fluorescence Correlative Microscopy Fluorescence correlation spectroscopy (FCS) experiments were performed using an MF-20 (Olympus, The assay was performed at room temperature on a 1000-well plate (Japan). Alexa Fluor 647-labeled myoglobin and Alexa Fluor 647-labeled myoglobin were used. The myoglobin-encapsulated micellar solution was placed in a pretreated 384-well glass-bottom microplate at 30 μL. To determine the structural parameters, a standard 63 μg / well with a molecular weight of 652 Da was added. A 3 nm solution (Olympus, Japan) was also placed in the plate. The sample was then illuminated with a 633 nm laser beam. The excitation was repeated 5 times for 20 seconds each. The data was then automatically fitted using the software. The fitting was performed using the fitting function.

[0064] 2. Results and Discussion 2.1. Synthesis and Chemical Analysis of Block Copolymers PEG-p(Lys-TFA) polymer is MeO-PEG-NH2

[20] The terminal primary amino group of The polymer was synthesized by ring-opening polymerization of Lys(TFA)-NCA. The results of the polymerized polymer were analyzed by GPC. As a result, the molecular weight distribution (M w / M n The protective group TFA was removed by alkaline hydrolysis (Fig. 11). After removal, the -OCH2CH2- (δ=3.5 ppm) of PEG and -C3H6 (δ=1.2 ppm~1.8 ppm) of p(Lys) were Using the roton ratio 1 The degree of polymerization (DP) of lysine was confirmed by H-NMR and found to be 37. Additionally, GPC (pH 3.3 acetate-buffered saline with 10 mM acetate containing 500 mM NaCl as the mobile phase; As a result of the analysis (flow rate: 0.75 mL / min), a single peak indicating a narrow molecular weight distribution was observed (Figure 12).

[0065] Next, CDM-Cl was reacted with the primary amine of PEG-p(Lys) to introduce CDM into the polymer. The peak intensity of -CH3 (δ = 2.0 ppm) on CDM was compared with the methylene peak on PEG and By comparing the β-, γ-, and δ-methylene protons of lysine, the amount of CDM introduced and The CDM introduction rate was confirmed. The CDM unit was calculated to be about 17, and the CDM addition rate was about 45%. Gp(Lys-CDM) was run in an acetate buffer solution at pH 3.3 (10 mM acetic acid with 500 mM NaCl) as the mobile phase. GPC using PEG-p(Lys-CDM) showed a narrow molecular weight distribution (Figure 13). This shows that the synthesis was successful with the quality required for micelle preparation.

[0066] 2.2. Core-crosslinked polyion complex (PIC) micelles without protein encapsulation (empty PIC micelles) Preparation of PEG-100 and its stability under various pH conditions PEG-p(Lys-CDM) has both amine moieties and amine-reactive CDM units, making it suitable for amine synthesis. The protonation of the amine and the formation of the CDM ring allow the free polymer to be obtained in an acidic pH environment. On the other hand, at near-neutral pH, the CDM group forms a stable amide bond with the amine, leading to further This generates carboxyl groups for the formation of polyion complexes (Scheme S2). Therefore, we investigated the PEG- The structure of p(Lys-CDM) was evaluated.

[0067] PEG-p(Lys-CDM) self-assembles into micelles at pH 7.4, which is higher than the other pH values. The derived count rate was determined by DLS and was found to be particles or high concentrations of particles

[24] The resulting micelles have a density of approximately 40 n at pH 7.4 (Fig. 2a). The count rate remained low at pH below 6.5. This indicates that PEG-p(Lys-CDM) did not associate with micelles. of polymer in hydrogenated phosphate buffer (10 mM) 1 PEG-p(Lys- The disappearance of the proton peaks originating from the polyamino acid and side chain structure of CDM was found, which indicates that The decrease in the mobility of the polyamino acid backbone due to the binding of the amine and CDM moieties is shown in Figure 14a. After adding the acid to the solution, the peaks from the polyamino acids and side chain structures were observed after 10 minutes of incubation. The recovery rate was up to 75% by incubation (Fig. 14b). This indicates that the release of polyamino acids under low pH conditions The pH-dependent micelle formation of PEG-p(Lys-CDM) was observed before the addition of protein. Care should be taken to avoid the formation of empty micelles.

[0068] The stability of self-assembled empty PIC micelles at pH 7.4 was investigated by exposing the micelles to solutions with different pH values. At pH 7.4, the size of empty PIC micelles decreased from 43 nm to 3 nm in 24 hours. The PDI fluctuation was small, with the count rate only decreasing by 20% (Fig. 3). On the other hand, at pH 6.5, empty PIC micelles are unstable and undergo a rapid decrease in size and count rate. The PDI increased by more than 0.4 during the first 5 hours of incubation (Figure 3). In 6.5, the measurement of micelle size after 5 hours was unreliable due to the high PDI and was omitted. These results indicate that empty PIC micelles collapse in a pH-responsive manner.

[0069] 2.3. In vitro cytotoxicity of PEG-p(Lys-CDM) against HEK293 cells PEG-p(Lys-CDM) can be safely used as a delivery vehicle for bioapplication of protein-encapsulating micelles. Therefore, we investigated the efficacy of PEG-p(Lys-CDM) in combination with HEK 293 cells. The cytotoxicity of PEG-p(Lys-CDM) was examined by culturing the cells for 48 hours. PEG-p(Lys-CDM) was used as a control because it is a precursor of PEG-p(Lys-CDM) and is widely used as a delivery vehicle. and used it.

[0070] As shown in Figure 4, PEG-(Lys-CDM) significantly improved cell proliferation compared to PEG-p(Lys) at all polymer concentrations. PEG showed low cytotoxicity and maintained cell viability of over 70% even at a polymer concentration of 1 mg / mL. The low toxicity of PEG-(Lys-CDM) is shown by DLS evaluation of PEG-(Lys-CDM) in DMEM (Figure 15). These results are likely due to the autonomous assembly of PEG- This indicates that p(Lys-CDM) is a highly safe delivery carrier.

[0071] 2.4. Preparation of protein-encapsulating micelles by precise pH control Proteins contain numerous negatively charged groups (glutamic acid, aspartic acid, and C-terminal carboxyl groups). heterocyclic amino acids with carboxyl groups and positively charged groups (lysine, arginine, and N-terminal amine) Therefore, PEG-p(Lys-CDM) is a macromolecule with a singly charged surface. The PIC is formed with the carboxyl group, and the pH-responsive CDM moiety binds to the primary amino acid in the protein. Furthermore, the amines in PEG-p(Lys-CDM) can be covalently bonded to the amine groups (Scheme S3). Further cross-linking of the micelle core by reacting with CDM groups that were not bound to the protein It is possible.

[0072] As observed above (Figure 2), PEG-p(Lys-CDM) autonomously forms micelles at medium pH. PEG-p(Lys-CDM) exists as a free polymer at pH 5, Prepare a polymer solution by dissolving PEG-p(Lys-CDM) in 10 mM acetate buffer (pH 5) and place it in an empty PIC microwell. Furthermore, the protein solution was prepared in 10 mM phosphate buffer (pH 8). The polymer solution was mixed with the lysine residue in PEG-p(Lys-CDM) to form a polyion complex. Self-assembly was performed via plex formation and amide formation with the CDM moiety. After mixing the and protein solutions, the pH was adjusted to 7.4. Free protein and micelles were analyzed by GPC. Since the myoglobin encapsulation efficiency was determined by GPC, the elution times of the myoglobin encapsulation efficiency were different. The globin was fluorescently labeled with Alexa Fluor 647 for fluorescence detection. The amount of protein absorbed was calculated by dividing the amount of protein added.

[0073] As shown in Table 1, myoglobin (a 17.6 kDa protein with an isoelectric point of 7) was purified with an efficiency of 62%. At 5 wt.%, it was encapsulated in micelles, resulting in micelles with a PDI of 0.18 and a size of 40 nm. Ultrafiltration using acid-buffered saline (pH 7.4, 10 mM phosphate buffer with 150 mM NaCl) After further purifying the micelles, the amount of myoglobin encapsulated per micelle was determined using FCS. The counts per molecule between the micelles and Alexa Fluor 647-labeled myoglobin were quantified. By calculating the ratio of the rates, approximately two Alexa Fluor 647-labeled myoglobin molecules were detected per micelle. Robin was confirmed to be encapsulated (Table 2).

[0074] [Table 1]

[0075] [Table 2]

[0076] In addition to myoglobin, bovine serum albumin (BSA) and lysozyme were also analyzed. Since the micelles have different sizes (molecular weights) and net charges (isoelectric points) from the bottles, the encapsulation capacity of the micelles was evaluated. As a result, it was found that PEG-p(Lys-CDM) could effectively incorporate these proteins into micelles. Furthermore, TEM observations showed that these protein-encapsulating micelles These results demonstrate the effectiveness of the present invention for encapsulating proteins. This shows that the micelle system has versatility.

[0077] S4. Encapsulation of different proteins into polymer micelles As shown in Table 3, PEG-p(Lys-CDM) binds various proteins with different molecular weights and isoelectric points (pI). Using the protein, micelles with a narrow particle size distribution could be formed.

[0078] [Table 3]

[0079] 2.5. Preparation of control myoglobin-encapsulated micelles To evaluate the effectiveness of the myo / m prepared in the previous section, control micelles without covalent bonds were constructed. To prepare the control micelles, we first added CDM slowly to the myoglobin solution. The CDM incorporation rate was measured by the fluorescamine method. The zeta potential of CC-myo was 92.8%, and the zeta potential of CC-myo was -29.5 mV, which was the same as that of native myoglobin. This indicates that the charge was converted by the introduction of CDM. In phosphate buffered saline (10 mM phosphate buffer containing 150 mM NaCl, pH 7.4), PEG-p(Lys) was mixed with CC-myo at an N / C (amino group / carboxyl group) ratio of 2:1 to form PIC myosin. As a control, a mixture of PEG-p(Lys) and native myoglobin was prepared in the same N / C medium as above. CC-myo formed PIC micelles with PEG-p(Lys) via electrostatic interactions ( Table 1). However, myoglobin without CDM modification did not form micelles with PEG-p(Lys). This is because the heterogeneous surface charge of myoglobin creates stable multi-ion complexes. [4] It is unfavorable to This is thought to be the reason.

[0080] 2.6. Micelle Stability The stability of the micelles was investigated using buffer solutions with different salt concentrations and pH. First, the pH stability test was carried out in 10 mM phosphate buffered saline (pH 6.5 or pH 7.4). The collapse of micelles (myo / m) composed of EG-p(Lys-CDM) and control micelles (CC-myo / m) was evaluated. The micellar size and PDI were measured every hour by DLS. and 6.5, the size and PDI remained unchanged, demonstrating high stability. CC-myo / m exhibited high stability at pH 7.4 as shown in Figure 5. On the other hand, at pH 6.5, CC-myo / m was rapidly destabilized. Furthermore, myo / m was salt-tolerant at both pH 6.5 and pH 7.4. While the empty PIC micelles collapsed rapidly under the same conditions (Fig. 5), the empty PIC micelles collapsed rapidly under the same conditions (Fig. 3). It was suggested that the protein stabilized the micelles of PEG-p(Lys-CDM).

[0081] The biological application of PIC micelles is due to the electrostatic interaction that maintains the micellar structure, which contributes to their retention in the blood. [25,26] Between Therefore, the electrostatic phase in the micelles is difficult to dissociate due to the high NaCl concentration (600 mM). interaction [25,27] completely inhibited, 10 mM phosphate buffer containing 600 mM NaCl at pH 7.4 or 6.5 Dialysis of the micelles using a 20,000 MWCO dialysis cassette under dilute conditions against 5 L of buffer. Stability was evaluated by taking samples over time and performing DLS analysis to follow the micelle stability. The control CC-myo / m, which was based solely on PIC, dissociated immediately after incubation in high salt. where myo / m is the size and count rate after 24 hours at pH 6.5 compared to pH 7.4. This suggests that rapid micelle disassembly occurs at acidic pathological pH. On the other hand, it has strong stability at physiological pH (Figure 6).

[0082] 2.7. Myoglobin release from myo / m The release of myo / m from micelles was measured in 5 L of 10 mM phosphate buffered saline at pH 7.4 and pH 6.5. The Alexa Fluor 647-labeled myo / m-encapsulated micelles were then dialyzed and evaluated. The fluorescence intensity of the micelles in the cassette was measured over time. At pH 7.4, myo / m was the ratio of the encapsulated micelle to the total micelle content. On the other hand, the release of myoglobin from the micelles was accelerated at pH 6.5. Approximately 70% of the encapsulated protein was released within 24 hours (Figure 7). The stability of the micelles and their rapid disintegration at pH 6.5 correlated with the pathological pH and ionic strength (150 This strongly suggests that micelles respond to the ion exchange reaction (i.e., to the ion exchange reaction) with α- and β-terminally bound α-terminally bound β ...

[0083] 2.8. Myoglobin activity Myoglobin oxygenation is measured by the Soret band (380-460 nm) and the Q band (480-650 nm). [22,28-3 0] Therefore, the myoglobin released from myo / m at pH 6.5 can be determined by the transfer of The activity of the amine was evaluated by UV / Vis spectroscopy. Upon addition of thorium, a Soret band appeared at 434 nm, which is the band of reduced myoglobin. Furthermore, the blue shift of the Soret band from 434 nm to 414 nm and the Q band A peak splitting of the band was observed after the introduction of O2, which is due to the oxygenated myoglobin [22,28,29] This corresponds to the band.

[0084] After that, when Ar gas was bubbled, the reverse changes in the Soret band and Q band appeared, indicating deoxidation. The release of myoglobin was confirmed (Fig. 8a). By alternating this process, the structure of myoglobin was successfully transformed into oxygenated and reduced forms. As a control, natural myoglobin was used (Fig. 8b, d). During the oxygenation or deoxygenation of myoglobin released from myoglobin or myo / m These results suggest that the proteins encapsulated in myo / m were released from their respective This indicates that the functionality of

[0085] 2.9. In vivo blood retention and biodistribution Many therapeutic proteins undergo aggregation in the blood and rapid renal excretion. [31,32] This causes retention in the blood In this example, PEG-p(Lys-CDM)-based micelles improved the pharmacokinetics of proteins. As a model protein to test its ability to improve blood flow,

[34] Myoglobin was used, which is known to be a marker for the chromatin. Myoglobin was fluorescently labeled with Alexa Fluor 647. The compound was photolabeled and encapsulated in micelles to investigate its in vivo blood retention and biodistribution.

[0086] Fluorescently labeled myo / m micelles showed a similar size distribution to unlabeled micelles. The blood retention of the micelles was recorded by real-time IV-CLSM. As shown in Figure 9a-c, In contrast, the covalently stabilized myo / m exhibited a half-life of over 120 minutes, whereas CC-myo / m (10 CC-myo / m and free myoglobin (9 min) showed short half-lives. Globin showed a strong fluorescent signal in the skin tissue, but myo / m did not extravasate into the skin. This indicates that the encapsulated myoglobin does not leak from the micelles in the blood. vinegar.

[0087] Next, myo / m and CC prepared from Alexa Fluor 647-labeled polymer and unlabeled myoglobin were analyzed. -myo / m was used to evaluate the in vivo blood retention and biodistribution of the polymer. The half-life of CC-myo / m was 120 minutes or more, similar to that of CC-myo / m. PEG-p(Lys) was detected in the blood within a few minutes. Because CC-myo / m is rapidly excreted from the blood, it is thought to be unstable in the blood. The half-life of fluorescently labeled myoglobin in CC-myo is approximately the same as that of myoglobin alone. This corresponds to the fact that the charge-converted myoglobin micelles rapidly dissociate in the blood (Fig. 9a, b). On the other hand, myo / m shows high stability in blood. This is because the blood retention of the fluorescently labeled polymer PEG-p(Lys-CDM) is longer than that of the fluorescently labeled protein (Figure 9c, e). This is to address the tendency of proteins to remain in the blood.

[0088] The biodistribution of myoglobin, CC-myo / m, and myo / m was compared with the main organs involved in the excretion of nanoparticles. The organs, namely kidney, liver and spleen, were evaluated 12 hours after administration. Cell nuclei were stained by tail vein administration of Hoechst 30 minutes before imaging. The kidneys, liver, and spleen were harvested and observed by ex vivo fluorescence imaging. As shown in Fig. 1, free myoglobin and CC-myoglobin show high accumulation in the kidney. This is consistent with the rapid excretion of free myoglobin and CC-myo / m from the blood. Compared with CC-myo / m and myoglobin, micelles suppressed accumulation in the kidney and did not accumulate in the liver. Ta.

[0089] Furthermore, CC-myo / m was tracked using Alexa Fluor 647-labeled PEG-p(Lys), and poly Due to the rapid excretion of the mer, almost no fluorescent signal was detected in the kidney, liver, and spleen. On the other hand, myo / m tracked using Alexa Fluor 647-labeled PEG-p(Lys-CDM) was The signal from α-myosin was mainly observed in the liver (Fig. 10e), which contains fluorescently labeled myoglobin. These results indicate the high stability of myo / m in blood. We have demonstrated the use of PEG-p(L) to prepare protein-loaded micelles for in vivo delivery. This shows that ys-CDM is useful.

[0090] 3. Conclusion The present inventors have developed a method for producing a tethered tether using a combination of a polyion complex and a pH-responsive amide bond. Using a novel polymer, PEG-p(Lys-CDM), which can encapsulate proteins, we have developed a method for encapsulating proteins. We have succeeded in developing pH-responsive polymeric micelles for this purpose. This indicates that these micelles are stable at pH 7.4 but rapidly disintegrate at pH 6.5. Furthermore, the micelles of the present invention encapsulating myoglobin were found to be free myoglobin and PIC-formed. Compared with micelles assembled solely by Furthermore, myoglobin released from micelles at pH 6.5 was similar to native myoglobin. It has been shown that the micelles of the present invention have the oxygenation and reduction ability of proteins. These findings demonstrate that the micelles of the present invention can target pathological tissues and As a protein nanocarrier effective for spatiotemporal control of protein activity in vivo, This shows the possibility of

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[0093] Example 2 1. Preparation of IL-12-encapsulated micelles In this example, IL-12-encapsulated micelles were prepared by precisely controlling the pH. Then, 2.5 mg of PEG-P(Lys-CDM) was dissolved in 0.5 mL of 20 mM phosphate buffer (pH 5), and the polymer The mixture was left to stand for 1 hour to prevent the self-association of IL-12 to form empty micelles. The IL-12 solution was dissolved in 0.5 mL of 20 mM phosphate buffer (pH 8). The solution was stirred (shaken) and then continuously stirred (shaken) for 6 hours. Then, 1 mL of buffer solution (pH 8) was added to the mixture, and the mixture was stirred (shaken) overnight.

[0094] The encapsulation efficiency was measured by ELISA. The concentration of free IL-12 in the mixture was measured as EL The amount of encapsulated IL-12 was calculated by detecting it with an ISA kit. As a result, the concentration of free IL-12 in 2 mL of the mixed solution was 1.6 μg / mL. The total concentration of IL-12 was Since the concentration was 5 μg / mL, the encapsulation efficiency was 68%.

[0095] 2. Purification and characterization of IL-12-encapsulated micelles The mixture was purified by dialysis. The mixture was loaded into a dialysis cassette with a MWCO of 100 kDa. The mixture was dialyzed overnight at 4°C against 0 mM phosphate buffer (pH 7.4) and 150 mM NaCl. The micellar solution was adjusted to a precise concentration for size and zeta potential measurements using a Zetasizer. The solution was finely adjusted (adjusted to have a polymer concentration of 1 mg / mL). As a result, the z-average size by DLS was 43 nm and the PDI was 0.229 (Figure 17). The surface of the filter was slightly negatively charged, with a zeta potential of -4.1±1.0 mV.

[0096] 3. In vitro drug release experiments In this section, the dialysis method was used again. The purified micelle solution was placed in a dialysis tube with a MWCO of 100 kDa. Load the set with 500 mL of 10 mM phosphate buffer (pH 7.4) + 150 mM NaCl, and 500 mL of 10 mM The samples were dialyzed against phosphate buffer (pH 6.5) + 150 mM NaCl at room temperature. Fluid was collected from the outside of the cassette and the IL-12 concentration in the sample was determined by ELISA. As a result, the micelles showed pH responsiveness. After 30 hours, the amount of IL-12 released at pH 6.5 was The amount released was approximately four times that at 7.4°C (Figure 18).

[0097] 4. In vitro cell experiments In this section, we investigated the physiological activity of the micelles and the released IL-12 from mouse splenocytes. The amount of INF-γ secreted was measured. 9-week-old BALB mice were sacrificed, and splenocytes were collected from the spleen. , 1 x 10 per well 5 The cells were seeded in a 96-well plate at a concentration of 0.01%. The micellar solution was diluted with buffer (p H 5), followed by ultracentrifugation of the external solution to adjust the concentration. The micelles and released IL-12 were then isolated at different concentrations. After 24 and 48 hours of incubation, each well was treated with IL-12. The supernatant was removed and the INF-γ concentration was measured using an ELISA kit.

[0098] As a result, after 24 hours, the IL-12 released from the IL-12-encapsulated micelles was significantly higher than that from the IL-12-encapsulated micelles. The increase in INF-γ concentration was suppressed, suggesting that micelle formation inhibits the binding of IL-12 to its receptor. The difference between released and native IL-12 was not significant (Figure 19). This indicates that micellization does not affect the physiological activity of the encapsulated protein. After a short time, the differences among the three groups decreased, a phenomenon attributed to the collapse of micelles.

Claims

1. A polymer complex comprising a protein and a block copolymer represented by the following formula (1): 【Chemistry 21】 [In the formula, R 1 and R 2 are each independently a hydrogen atom or an optionally substituted carbon atom A linear or branched alkyl group having 1 to 12 carbon atoms, or an azide, amine, maleimide, or ligno represents a band or labeling agent, R 3 represents a compound represented by the following formula (I): 【Chemistry 22】 (In the formula, R a and R b are each independently a hydrogen atom or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group , a heterocyclic alkyl group, a hydroxy group, an alkoxy group, or an aryloxy group. Ta, R a and R b are bonded to each other and form an aromatic ring or a cycloalkyl ring together with the carbon atoms to which they are bonded. An alkyl ring may be formed. a and R b The bond between the carbon atoms to which , may be a single bond or a double bond.) L 1 is NH, CO, or the following formula (11): -(CH 2 ) p1 -NH- (11) (wherein p1 represents an integer of 1 to 6.) or a group represented by the following formula (12): -L 2a -(CH 2 ) q1 -L 3a - (12) (In the formula, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a NH or represents CO. q1 represents an integer from 1 to 6. represents a group represented by m1 and m2 each independently represent an integer of 0 to 500 (provided that the sum of m1 and m2 is 10 to 500). m3, m4 and m5 each independently represent an integer of 1 to 5, and n represents an integer of 0 to 500. Represents an integer. The symbol " / " indicates that the (m1 + m2) monomer units on either side of it can be arranged in any order. It means that.]

2. The compound represented by formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig): The complex of claim 1 , wherein the complex is one of: 【Chemistry 23】

3. The compound represented by formula (I) is a compound represented by the following formula (Ia) or (Ib): The described complex. 【Chemistry 24】

4. 2. The block copolymer according to claim 1, wherein the block copolymer represented by formula 1 is represented by the following formula (2): A complex of. 【Chemistry 25】

5. 10. The method of claim 1, wherein the protein is covalently attached to a block copolymer of formula 1. A complex of.

6. The complex of claim 5 , wherein the covalent bond is cleaved in a pH-dependent manner.

7. A method for the preparation of a polymer conjugate according to any one of claims 1 to 6, comprising administering to a subject on a cell surface, in a cell, or in a cell. A protein delivery device chosen from extracellular to extracellular.

8. A block copolymer represented by the following formula (1) is selected from the cell surface, intracellular and extracellular regions. Protein delivery kit to either. 【Chemistry 26】 [In the formula, R 1 and R 2 are each independently a hydrogen atom or an optionally substituted carbon atom A linear or branched alkyl group having 1 to 12 carbon atoms, or an azide, amine, maleimide, or ligno represents a band or labeling agent, R 3 represents a compound represented by the following formula (I): 【Chemistry 27】 (In the formula, R a and R b are each independently a hydrogen atom or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group , a heterocyclic alkyl group, a hydroxy group, an alkoxy group, or an aryloxy group. Ta, R a and R b are bonded to each other and form an aromatic ring or a cycloalkyl ring together with the carbon atoms to which they are bonded. An alkyl ring may be formed. a and R b The bond between the carbon atoms to which , may be a single bond or a double bond.) L 1 is NH, CO, or the following formula (11): -(CH 2 ) p1 -NH- (11) (wherein p1 represents an integer of 1 to 6.) or a group represented by the following formula (12): -L 2a -(CH 2 ) q1 -L 3a - (12) (In the formula, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a NH or represents CO. q1 represents an integer from 1 to 6. represents a group represented by m1 and m2 each independently represent an integer of 0 to 500 (provided that the sum of m1 and m2 is 10 to 500). represents an integer; m3, m4, and m5 each independently represent an integer of 1 to 5; and n is an integer of 0 to 500. Represents. The symbol " / " indicates that the (m1 + m2) monomer units on either side of it can be arranged in any order. It means that.]

9. The compound represented by formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig): The kit according to claim 8, wherein the kit further comprises one of the following: 【Chemistry 28】

10. The compound represented by formula (I) is a compound represented by the following formula (Ia) or (Ib): The kit described. 【Chemistry 29】

11. 9. The block copolymer according to claim 8, wherein the block copolymer represented by formula 1 is represented by the following formula (2): Kit. 【Transformation 30】

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