Title of invention: Protein-encapsulating polymer micelle
Technical field
[0001]
　The present invention relates to protein-encapsulating polymer micelles that can improve stability in harsh in vivo environments by using block copolymers. All disclosures of cited references are incorporated herein by reference in their entirety.
Background technology
[0002]
Since proteins are bioactive substances that exist everywhere in the body, they are used for the treatment of various intractable diseases such as cancer, autoimmune diseases, and metabolic disorders. However, since systemic administration of a protein alone is subject to enzymatic degradation and renal excretion and is immunogenic, it is necessary to develop a delivery carrier for biological application of the protein. Therefore, the development of a protein-PEG conjugate in which a biocompatible polymer poly (ethylene glycol) (PEG) is introduced into a protein is underway, and the suppression and size of interaction with proteolytic enzymes and immune cells are being promoted. It is possible to overcome protein challenges [1-4] by increasing the amount. In fact, many protein-PEG conjugates are FDA approved and have a multi-billion dollar market [5,6] for protein formulations. However, while protein PEGylation suppresses enzymatic degradation, renal excretion, and immunogenicity [7,8] , protein inactivation and protein function due to irreversible chemical modification of the protein [6,9]. There are issues such as inadequate spatiotemporal control. Therefore, the development of a delivery carrier capable of specifically releasing a target tissue [10] while suppressing the expression of the protein in a normal tissue by sawing the protein through a reversible chemical bond has been promoted. There is.
[0003]
　Stimulation-responsive nanocarriers can be specifically released to the target tissue while preserving the activity of the protein by sensing the bioactive substance in the target tissue [4,11] . Among such nanocarriers, core-shell polymer micelles formed by autonomous association of block copolymers and proteins are external by introducing environment-responsive sites into the core-forming chains of block copolymers. It can induce the release of proteins in response to stimuli [4] . External stimuli to which macromolecular micelles can respond include pH, for example, many diseases (eg, cancer or autoimmune diseases) have a lower pH (pH 6.5) than normal tissue (pH 7.4) [12,13]. ~ 7.2) is shown.
[0004]
　On the other hand, the present inventor has so far added a PEG-polycation to a protein in which an amino group is converted into a carboxyl group by a pH-responsive maleic anhydride derivative [14-16] to obtain a polyion complex (PIC) type height. It was shown that molecular micelles can be prepared. These micelles stably encapsulate the protein in the core at the pH of normal tissue (pH 7.4), but at acidic pH (pH 6.5-7.2) in the target tissue, the pH-responsive maleic anhydride derivative is cleaved to cleave the protein. Succeeded in releasing.
　However, for medical applications, it is important to increase the accumulation in target tissues by improving the retention in blood.
Outline of the invention
Problems to be solved by the invention
[0005]
　Therefore, in order to increase the therapeutic effect of therapeutic proteins, it is important to develop micelles that enable increased blood retention and efficient release of proteins under acidic conditions.
Means to solve problems
[0006]
　The present invention introduces a pH-responsive maleic anhydride derivative into the core-forming chain of a block copolymer to form a reversible covalent bond with the amino group of the protein, thereby increasing the stability of the micelles and under acidic conditions. Aimed at efficient protein release in. We also aimed to further stabilize micelles by forming PICs between the amino group of the core-forming chain of the block copolymer and the carboxyl group of the protein. The purpose is to stabilize the micelle structure by covalent bond and PIC formation, and to increase the retention in blood.
[0007]
　That is, the present invention is as follows.
[1] A polymer complex containing a protein and a block copolymer represented by the following formula (1).
[Chemical formula 1]

[In the formula, R 1 and R 2 are each independently a hydrogen atom, or a linear or branched alkyl group having 1 to 12 carbon atoms which may be substituted, or an azide. Representing an amine, maleimide, ligand or labeling agent,
　R 3 represents a compound represented by the following formula (I), and
[Chemical formula 2]

(in the formula, R a and R b are independently hydrogen atoms, respectively. Alternatively, it represents an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group which may be substituted. A and R b may be bonded to each other to form an aromatic ring or a cycloalkyl ring together with the carbon atom to which each is bonded . The bond between the carbon atoms to which R a and R b are bonded, respectively, may be formed. , It may be a single bond or a double bond.)
　L 1 is NH, CO, or the group represented by the following equation (11):-(
　　CH 2 ) p1 -NH- (11)
(in the equation, p1 represents an integer from 1 to 6)
, or the following equation . (12):
　　-L 2a- (CH 2 ) q1 -L 3a- (12)
(In the equation, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a is NH or COO. Represents CO. Q1 represents an integer from 1 to 6)
, and
　m1 and m2 independently represent integers from 0 to 500 (provided that the total 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.
　The notation of "/" indicates that the sequence order of each of the (m1 + m2) monomer units shown on the left and right of the "/" is arbitrary. ]
[2] The complex according to [1], wherein the compound represented by the formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig).
[Chemical

formula 3] The complex according to [2], wherein the compound represented by the formula (I) is a compound represented by the following formula (Ia) or (Ib).
[Chemical formula 4]

[4] The complex according to [1], wherein the block copolymer represented by the formula 1 is represented by the following formula (2).
[Chemical formula 5]

[5] The complex according to [1], wherein the protein is covalently bonded to the block copolymer represented by the formula 1.
[6] The complex according to [5], wherein the covalent bond is cleaved in a pH-dependent manner.
[7] A protein delivery device selected from cell surface, intracellular and extracellular, comprising the polymer complex according to any one of [1] to [6].
[8] A protein delivery kit to any of cell surface, intracellular and extracellularly selected, comprising the block copolymer represented by the following formula (1).
[Chemical formula 6]

[In the formula, R 1 and R 2 are independent hydrogen atoms, or linear or branched alkyl groups having 1 to 12 carbon atoms which may be substituted, or azides. Represents an amine, maleimide, ligand or labeling agent,
　R 3Represents a compound represented by the following formula (I),
[Chemical formula 7]

(in the formula, R a and R b are each independently hydrogen atom or optionally substituted alkyl group, alkenyl group, It represents a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group. In addition, R a and R b are bonded to each other and each is bonded. An aromatic ring or a cycloalkyl ring may be formed together with the carbon atoms forming . The bond between the carbon atoms to which R a and R b are bonded may be a single bond or a double bond. It may be.)
　L 1 is indicated by NH, CO, or the following equation (11) :-(
　　CH 2 ) p1 -NH- (11)
(in the equation, p1 represents an integer from 1 to 6)
. Or the following equation (12):
　　-L 2a- (CH 2 ) q1 -L 3a- (12)
(In the equation, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, L 3a represents NH or CO. Represents
the group indicated by ) ,
　m1 and m2 independently represent integers from 0 to 500 (however, the sum of m1 and m2 represents an integer from 10 to 500), and m3, m4 and m5 represent. Each independently represents an integer from 1 to 5, and n represents an integer from 0 to 500.
　The notation of "/" indicates that the sequence order of each of the (m1 + m2) monomer units shown on the left and right of the "/" is arbitrary. [9 ]
The kit according to [8], wherein the compound represented by the formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig).
[Chemical formula 8]

The kit according to [9], wherein the compound represented by the formula (I) is a compound represented by the following formula (Ia) or (Ib).
[Chemical Formula 9]

[11] The kit according to [8], wherein the block copolymer represented by the formula 1 is represented by the following formula (2).
[Changing 10]

A brief description of the drawing
[0008]
FIG. 1 shows pH-responsive protein-encapsulating micelles due to polyion complex formation and pH-responsive amide binding.
FIG. 2 shows self-assembly of PEG-p (Lys-CDM) in buffers showing different pH. a) A derived count rate normalized by the PEG-p (Lys-CDM) count rate at pH 7.4. 1 mg of PEG-p (Lys-CDM) in 10 mM acetate buffer (pH 4 or pH 5) containing 150 mM NaCl or 10 mM phosphate buffer (pH 6.5 or pH 7.4) containing 150 mM NaCl. After adding at a concentration of / mL and vortexing for 1 minute, the mixture was incubated for 1 hour and DLS measurement was performed. The data are shown as mean ± standard deviation (n = 3). b) Particle size distribution of Empty-PIC micelles (empty micelles) formed at pH 7.4.
FIG. 3 is a diagram showing the stability of empty micelles prepared in a pH 7.4 buffer. Empty micelles were added to 10 mM phosphate buffer (pH 6.5 (gray dots) or 7.4 (black dots)) containing 150 mM NaCl to adjust the final concentration to 0.5 mg / mL and DLS measurements were performed. a) is the particle size, b) is the PDI, and c) is the count rate standardized by the count rate before dilution.
FIG. 4 shows in vitro cytotoxicity of PEG-p (Lys-CDM) (gray line) to HEK 293 cells (after cells have been cultured for 48 hours at different polymer concentrations). PEG-p (Lys) (black line) was used as a control. The data are shown as mean ± standard deviation (n = 4).
FIG. 5 shows the stability of protein-encapsulating micelles in solutions showing different pH. Particle size (a) and PDI of myo / m (gray circle and black circle) and CC-myo / m (white circle) in 10 mM phosphate buffer at pH 6.5 (gray line) and pH 7.4 (black line) (b).
FIG. 6 shows the stability of myo / m after dilution with 10 mM phosphate buffer containing 600 mM NaCl, which shows different pH. Particle size (a) of myo / m at pH 6.5 (gray line) and pH 7.4 buffer (black line), and normalized count rate (b). It can be seen that myo / m collapsed in a pH 6.5 buffer solution.
FIG. 7 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.
FIG. 8 is a diagram showing an evaluation of myoglobin activity. a) UV / Vis absorption spectra of oxygen-type myoglobin (gray line) after introduction of O 2 gas and reduced myoglobin (black line) after introduction of Ar gas. Insertion: Spectrum of myoglobin emitted from micelles at 500-600 nm. b) UV / Vis absorption spectra of natural oxygen-type myoglobin (gray line) after introduction of O 2 gas and reduced myoglobin (black line) after introduction of Ar gas. Inset: Spectrum of natural myoglobin at 500-600 nm. cd) O 2(Square mark) / Ar (Triangle mark) Shows the absorbance of released myoglobin (c, white mark) and natural myoglobin (d, black mark) at 414 nm when gas is introduced alternately.
FIG. 9 is a diagram showing blood retention of fluorescently labeled myoglobin, CC-myo / m and myo / m measured by IV-CLSM. a)-c) Alexa Fluor 647 labeled myoglobin (red), a) myoglobin alone, b) CC-myo / m and c) myo / m. d) -e) CC-myo / m and e) myo / m prepared using Alexa Fluor 647 labeled polymer (red). Normalization and quantification were performed using the fluorescence intensities in the veins (red trapezoid) and skin (green trapezoid) of the microscopic images (a to e, left panel) immediately after administration of the sample (a to e, right panel).
FIG. 10 shows the microdistribution of fluorescently labeled myoglobin, CC-myo / m and myo / m in the kidney, liver and spleen. a)-c) Prepared with Alexa Fluor 647 labeled myoglobin (red), a) myoglobin alone, b) CC-myo / m, c) myo / m. d) -e) CC-myo / m, e) myo / m prepared using Alexa Fluor 647 labeled polymer (red). Cell nuclei were stained with Hoechst (cyan). Scale bar: 100 μm.
[Fig. 11] Fig. 11 is a diagram showing a chemical analysis of PEG-p (Lys-TFA). a) 1 of PEG-p (Lys-TFA) in DMSO-d 6H-NMR spectrum, b) GPC chromatogram of PEG-p (Lys-TFA), showing a monomodal peak and a narrow molecular weight distribution (Mw / Mn = 1.03). (Flow rate: 0.8 mL / min, mobile phase: DMF solution containing 10 mM LiCl).
[Fig. 12] Fig. 12 is a diagram showing a chemical analysis of PEG-p (Lys). a) 1 H-NMR spectrum of PEG-p (Lys) at D 2 O , b) GPC chromatogram of PEG-p (Lys). (Flow velocity: 0.75 mL / min, mobile phase: 10 mM acetate and 500 mM NaCl acetate buffered saline (pH 3.3)) [Fig. 13] Fig. 13 shows the characteristics of PEG-p (Lys-CDM). a) 1 H-NMR spectrum of PEG-p (Lys-CDM) in DMSO-d 6 , b) Aqueous phase GPC chromatogram of PEG-p (Lys-CDM). (Flow velocity: 0.75 mL / min, eluent: 10 mM acetate and 500 mM NaCl acetate buffered saline (pH 3.3)) [Fig. 14] Fig. 14 showing the characteristics of PEG-p (Lys-CDM). a) 1 of PEG-p (Lys-CDM) in 10 mM deuterated phosphate buffer (0.70 ml)

1 NMR spectrum (25 ° C, pH 7.4). It is believed that the proton-derived peak intensities of polyamino acids were lower than expected from the PEG proton-derived peaks due to the limited mobility of protons in the polymer due to micelle formation. b) 1 H-NMR spectrum of PEG-p (Lys-CDM) after adding 2 M heavy hydrochloric acid (volume ratio 1:35) and incubating for 10 minutes. The acid treatment restored the proton-derived peak intensity of the polyamino acid by about 75%, suggesting that the disintegration of micelles under acidic conditions increased the mobility of protons in the polymer.
FIG. 15 is a diagram showing the size distribution of 1 mg / mL PEG-p (Lys-CDM) in DMEM.
FIG. 16 is a TEM image of lysozyme (left), myoglobin (center) and BSA-encapsulated micelles (right). Scale bar: 50 nm. The morphology of the micelles was observed by TEM (JEM-1400, JEOL). Protein-encapsulating micelles were stained with phosphotungstic acid (PTA) (2%, w / v) and placed on a 400 mesh copper grid. The image was taken at a magnification of 50,000 times.
[Fig. 17] Fig. 17 is a diagram showing the size distribution of micelles of IL-12 encapsulated micelles.
FIG. 18 is a diagram showing the release of IL-12 from IL-12 encapsulated micelles.
FIG. 19 is a diagram showing the amount of INF-γ secretion in mouse splenocytes by IL-12-encapsulating micelles.
Embodiment for carrying out the invention
[0009]
　Therapeutic proteins are expected for the treatment of intractable diseases, but there are various challenges with respect to their systemic administration, such as instability, short half-life, and non-specific immune response. Therefore, an approach of delivering proteins by stimulus-responsive nanocarriers can be an effective strategy for tissue-selectively enhancing protein activity in target tissues. In the present invention, in order to release the loaded protein depending on pH, a polyion complex is formed between the protein and the block copolymer, and the protein is formed through a covalent bond that can be cleaved under predetermined pH conditions. We have developed a polymer micelle that has the ability to encapsulate.
[0010]
　The carboxydimethylmaleic anhydride (CDM) -amide bond is stable at physiological pH (pH 7.4) but is cleaved at pH 6.5, the pathophysiological pH of tumor and inflamed tissue. Therefore, CDM was selected as the pH-responsive functional group. In the present invention, by using a poly (ethylene glycol) -poly (L-lysine) block copolymer having a CDM addition of 45% or more, 50% or more can be added to different proteins having various molecular weights and isoelectric points. It was included with efficiency. Using myoglobin-encapsulated micelles (myo / m) as a model confirmed the stability of micelles under physiological conditions, as well as the disintegration of micelles and the release of functional myoglobin at pH 6.5. Furthermore, myo / m had an improved half-life in blood compared to myoglobin alone and micelles associated only by electrostatic interactions without covalent bonds. Therefore, the above model demonstrated the usefulness of the system for in vivo delivery of therapeutic proteins.
[0011]
　CDM was selected as the pH responsive site in the present invention because the CDM-amide bond is unstable at pH 6.5 [17-19] , thereby allowing the release of conjugated amino compounds at pathophysiological pH. .. Therefore, the resulting protein-encapsulating micelles form stable cross-linked cores at physiological pH, but decompose into free block copolymers and active proteins at pH 6.5 (Fig. 1). Therefore, in the present invention, the ability of these micelles to contain various proteins was evaluated. Furthermore, the present inventor used micelles containing myoglobin and IL-12 as a model for in vitro stability of micelles, protein release at different pH, and in vivo blood retention after systemic administration. evaluated.
[0012]
1. 1. Polymer complex of the
　present invention The polymer composite of the present invention is a protein-encapsulating polymer micelle complex (polyion complex: PIC), and is a specific cationic polymer (block copolymer (also referred to as block copolymer), graft copolymer, etc.). ) And the protein (details of the protein will be described later).
[0013]
(1) Cationic Polymer
　The specific cationic polymer which is a component of the PIC of the present invention is a cationic polymer having a polycation portion at least partially. The cationic polymer may be, for example, a block copolymer or a graft polymer having a polyethylene glycol (PEG) moiety and a polycation moiety, and is not limited. A preferred embodiment can be appropriately selected depending on the intended use of the PIC of the present invention.
[0014]
　The structure (for example, degree of polymerization, etc.) of the PEG and polycation is not limited, and any structure can be selected. Among them, the polycation is preferably a polypeptide having a cationic group in the side chain. The term "cationic group" as used herein means not only a group to which a hydrogen ion is coordinated to become a cation, but also a group to which a hydrogen ion is coordinated to become a cation. All known cationic groups are included. A polypeptide having a cationic group in the side chain is a peptide bond of a known amino acid (lysine, arginine, histidine, etc.) having a basic side chain, or a peptide bond of various amino acids to the side chain (the side chain). For example, a side chain of aspartic acid or glutamic acid) is substituted so as to have a cationic group.
[0015]
　Specific examples of the specific cationic polymer include block copolymers represented by the following general formula (1).
[Chemical 11]

[0016]
　Here, in the structural formula of the general formula (1), the block portion in which the number of repeating units (degree of polymerization) is n is the PEG portion, and the block portion in which the portion in which the number of repeating units is m1 and the portion in m2 are combined (general). In equation (1), the part shown in [] is the polycation part. Further, the notation of "/" in the structural formula of the polycation portion means that the arrangement order of each monomer unit shown on the left and right thereof is arbitrary. For example, when the block portion composed of the monomer units A and B is described as [-(A) a-/-(B) b-], a block portion consisting of a A and b B is used. This means that each of the total (a + b) monomer units may be randomly linked in any order (provided that all A and B are linearly linked. ).
[0017]
　In the general formula (1), R 1 and R 2 are independently hydrogen atoms, or linear or branched alkyl groups having 1 to 12 carbon atoms which may be substituted, or azides and amines. Represents a functional group such as maleimide, ligand or labeling agent.
　Examples of the linear or branched alkyl group having 1 to 12 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group and a tert-butyl group. , N-pentyl group, n-hexyl group, decyl group, undecyl group and the like. Examples of the substituent of the alkyl group include an acetalized formyl group, a cyano group, a formyl group, a carboxyl group, an amino group, an alkoxycarbonyl group having 1 to 6 carbon atoms, an acylamide group having 2 to 7 carbon atoms, and a siroxy group. , Cyrilamino group, trialkylsiloxy group (each alkylsiloxy group has 1 to 6 carbon atoms independently) and the like.
[0018]
The ligand molecule means a compound used for the purpose of targeting a specific biomolecule, and examples thereof include antibodies, aptamers, proteins, amino acids, small molecule compounds, and monomers of biopolymers. Examples of the labeling agent include fluorescent labeling agents such as rare earth fluorescent labeling agents, coumarin, dimethylaminosulfonylbenzoxadiazole (DBD), dancil, nitrobenzoxadiazole (NBD), pyrene, fluorescein, and fluorescent proteins. , Not limited to these.
[0019]
　If the substituent is an acetal-protected formyl group, this substituent is converted to another substituent, the formyl group (or aldehyde group; -CHO), when hydrolyzed under mild acidic conditions. can do. In addition, when the above-mentioned substituent (particularly the substituent in R 1 ) is a formyl group, or a carboxyl group or an amino group, for example, an antibody or a fragment thereof or other functional or targeting directivity is obtained via these groups. It is possible to bind a possessing protein or the like.
[0020]
　In the general formula (1), R 3 represents the compound represented by the following general formula (I).
[Chemical formula 12]

　In the above formula (I), R a and R b are independently hydrogen atoms or optionally substituted alkyl groups, alkoxy groups, cycloalkyl groups, aryl groups, aralkyl groups, and acyls. Represents a group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group. Further, R a and R b may be bonded to form an aromatic ring or a cycloalkyl ring together with the carbon atom to which each is bonded. Further, in the formula (I), the bond between the carbon atoms to which R a and R b are bonded may be a single bond or a double bond, and is not limited. In the formula (I), in order to show both bonding modes together, the carbon atoms are represented by one solid line and another broken line.
[0021]
　L 1 is NH, CO, the following general formula (11) :-(
　　CH 2 ) p1 -NH- (11)
(in the formula, p1 represents an integer from 1 to 6)
, or the following general Equation (12):
　　-L 2a- (CH 2 ) q1 -L 3a- (12)
(In the equation, L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a is NH. Or it represents CO. Q1 represents an integer from 1 to 6)
.
[0022]
　In the above equation (1), m1 and m2 independently represent integers from 0 to 500 (however, the sum of m1 and m2 represents an integer from 10 to 500), and m3, m4 and m5 represent, respectively. Independently represents an integer from 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 to 45,000, and more preferably 28,000 to 34,000. For each block portion, the molecular weight (Mw) of the PEG moiety is preferably 8,000 to 15,000, more preferably 10,000 to 12,000, and the molecular weight (Mn) of the polycation moiety is 15,000 to 15,000 as a whole. It is preferably 30,000, more preferably 18,000 to 22,000.
[0024]
　The method for producing the cationic polymer represented by the general formula (1) is not limited, but for example, a segment (PEG segment) containing R 1 and a block portion of the PEG chain is synthesized in advance, and this PEG segment is used. A method of polymerizing a predetermined monomer in order at one end (the end opposite to R 1 ) and then substituting or converting the side chain so as to contain a cationic group, or the above PEG segment and a cation. Examples thereof include a method in which a block portion having a side chain containing a sex group is synthesized in advance and these are linked to each other. The methods and conditions of various reactions in the production method can be appropriately selected or set in consideration of the conventional method.
[0025]
　In one embodiment of the present invention, the compound represented by the formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig).　
[Chemical 13]

[0026]
　In a preferred embodiment of the present invention, the compound represented by the formula (I) is a compound represented by the following formula (Ia) or (Ib).
[Changing 14]

[0027]
　In formula (I), the substituent is a saturated or unsaturated acyclic or cyclic hydrocarbon group. In the case of an acyclic hydrocarbon group, it may be linear or branched. Hydrocarbon groups include, for example, C 1 - C 20 alkyl group, C 2 -C 20 alkoxy group , C 4 -C 20 cycloalkyl group, C 6 -C 18 aryl group, C 6 -C 20 aralkyl group, C 1 Examples include -C 20 alkoxy group and C 6 -C 18 aryloxy group.
[0028]
　The compound represented by the formula (I) is used as a charge regulator. The compound represented by the formula (I) converts the charge of the basic or neutral protein as a whole into the charge of the acidic protein. In other words, the charge regulator of the present invention controls the amount of charge so that the protein whose total charge is on the positive (+) side or the neutral state is the protein whose total charge is on the negative (-) side. Therefore, it can be said that the conversion of the total charge is performed. Specifically, the conversion of the total charge is such that the compound represented by the above formula (I) or a derivative thereof binds to an amino group (a group having a positive charge) contained in the protein and makes the entire protein negatively charged. It is done by. For this purpose, the bond has, for example, a structure such that the compound represented by the above formula (I) and an amino group in a protein are bonded (covalently bonded) and represented by the following formula (I'). It is done by taking.
[Changing 15]

[0029]
　Regarding the above-mentioned binding, for example, when the compound represented by the above-mentioned formula (I) is a compound represented by the above-mentioned formulas (Ib) and (Ic), the structure represented by the above-mentioned formula (I') after the binding is as follows. , It becomes as follows.
[Changing 16]

　
[0030]
　In a further aspect of the present invention, the block copolymer represented by the formula 1 is represented by the following formula 2.
[Changing 17]

　
[0031]
(2) Protein
　In the PIC of the present invention, the protein that is a constituent of the core portion may be a protein (charge conversion protein) whose overall charge is converted by the compound represented by the above-mentioned formula (I). Specifically, it may be a protein whose total charge is converted from the total charge of the basic or neutral protein (plus side or neutral state) to the minus side like the total charge of the acidic protein. .. It can be said that the protein converted so that the total charge is on the negative side is an anionic substance (polyanion) as a whole protein. Therefore, by electrostatic interaction with the polycationic moiety in the cationic polymer, it is possible to easily form a micelle-like complex that was originally difficult to form with a basic or neutral protein.
[0032]
　The type of protein used in the present invention is not limited as long as it is originally contained in a basic or neutral protein. The protein used in the present invention includes not only simple proteins but also glycoproteins, lipid proteins and the like. Further, the protein used in the present invention is not limited to a protein having a full-length amino acid sequence, but also includes a partial fragment thereof, a peptide, and the like, and further, a protein consisting of two or more molecules (dimers), a partial sequence thereof, or a full-length protein. It also includes fusion proteins between sequences. Further, the protein used in the present invention is not limited to those composed of natural amino acids, and also includes modified proteins containing at least a part of unnatural amino acids as constituents. Further, the protein used in the present invention also includes a protein to which various labeling substances and the like are added as appropriate. Specific examples of the protein used in the present invention include, but are limited to, hem proteins, various cytokines, various enzymes, antibodies (for example, antibodies against nuclear pore complexes, etc.) or antibody fragments. It's not a thing.
[0033]
(3) Polyion complex (PIC) In
　the PIC of the present invention, a protein and a part of the above-mentioned cationic polymer (polycation part) electrostatically interact with each other to form a core part, and the cation is formed. It can be said to be a core-shell type micelle-like complex in which other parts (parts including the PEG part) in the sex polymer form a shell part around the core part.
[0034]
　The PIC of the present invention can be easily prepared, for example, by mixing a protein and a cationic polymer in an arbitrary buffer (for example, Tris buffer). The mixing ratio of the cationic polymer and the protein is not limited, but in the present invention, for example, the total number of cationic groups (for example, amino groups) in the block copolymer (N) and the total number of carboxyl groups in the protein are not limited. The ratio (N / C ratio) to (C) can be 0.1 to 200, 0.5 to 100, and further 1 to 50. When the N / C ratio is in the above range, it is preferable in that free cationic polymers can be reduced. The cationic group (N) means a group capable of forming an ionic bond by electrostatic interaction with a carboxyl group in a protein contained in a micelle.
[0035]
　The size of the PIC of the present invention is not limited, but for example, the particle size according to the dynamic light scattering measurement method (DLS) is preferably 5 to 200 nm, and more preferably 10 to 100 nm.
[0036]
　本発明のPICは、細胞内に導入された後、内包していたタンパク質を放出するが、この際、細胞質内におけるpH環境の変化（弱酸性環境下（例えばpH 5.5程度）に変化）により、前記式（I）で示される化合物が当該タンパク質から解離する（結合が切れる）。これにより、当該タンパク質の全体としての電荷（総荷電）が、当該タンパク質がもともと有する固有の電荷（総荷電）に回復するため、導入した細胞内においては、当該タンパク質をその構造及び活性等が再生した状態で存在させることができる。
[0037]
２．タンパク質送達デバイス
　本発明においては、上述したポリイオンコンプレックス（PIC）を含むタンパク質送達デバイスが提供される。本発明のタンパク質送達デバイスは、細胞内外の酸化還元環境の変化を利用し、PICのコア部分に内包した所望のタンパク質（電荷変換タンパク質）を、標的細胞の細胞表面、細胞内及び細胞外から選ばれるいずれかに効率的に導入する手段として使用できる。
[0038]
　Specifically, a solution containing a PIC containing a desired protein is administered to a test animal to be taken up by a target cell in the body. After that, when the PIC taken up into the cell reaches the endosome, the compound represented by the formula (I) is desorbed from the protein, and the charge balance in the PIC is changed, so that the PIC is disrupted. When the PIC collapses, the protein is released from the PIC and at the same time the polymer dissociated from the PIC damages the endosomal membrane. This destroys the endosomes, thus achieving delivery of the released protein into the cytoplasm.
　For example, in the case of micelles containing cytokines such as IL-12, the protein is released extracellularly and the protein binds to a receptor on the cell surface, so that the cell surface can be targeted for delivery. When an enzyme that expresses its function in the cell is delivered by a micelle, the protein is released inside the cell and the enzyme functions, so that the intracellular can be targeted for delivery. When delivering an antibody, the extracellularly secreted protein may be targeted, so that the extracellular can be targeted for delivery. Of course, it is also possible to target two or three combinations of cell surface, intracellular and extracellular.
[0039]
　The protein delivery device of the present invention can be applied to various mammals such as humans, mice, rats, rabbits, pigs, dogs and cats, and is not limited thereto. As the method of administration to the test animal, a parenteral method such as intravenous drip infusion is usually adopted, and each condition such as the dose, the number of administrations and the administration period may be appropriately set according to the type and condition of the test animal. can.
[0040]
　The protein delivery device of the present invention can be used for a treatment (for example, enzyme replacement therapy, immunotherapy using an antibody, etc.) for introducing a desired protein into cells causing various diseases. Therefore, the present invention relates to a pharmaceutical composition containing the above-mentioned PIC (for example, a pharmaceutical composition for enzyme replacement therapy or immunotherapy), and a method for treating various diseases using the above-mentioned PIC (for example, enzyme replacement therapy or the like). , Immunotherapy using antibodies, etc.) can also be provided. The administration method and conditions are the same as described above.
[0041]
　For the above pharmaceutical compositions, excipients, fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, dissolution aids commonly used in drug production Agents, preservatives, flavoring agents, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and used, and can be prepared by a conventional method. In addition, the form of the pharmaceutical composition is usually an intravenous injection (including an infusion), and is provided, for example, in the form of a unit dose ampoule or a multi-dose container.
[0042]
3. 3. Protein Delivery Kit The protein delivery kit
　of the present invention is characterized by containing the block copolymer. The kit can be preferably used for various therapeutic methods using desired proteins such as enzyme replacement therapy and immunotherapeutic methods using antibodies.
[0043]
　In the kit of the present invention, the storage state of the cationic polymer is not limited, and a solution state or a powder state can be selected in consideration of its stability (preservability), ease of use, and the like. The kit of the present invention may contain other components other than the block copolymer. Examples of other components include various buffers, various proteins to be introduced into cells (charge conversion proteins), lysis buffers, instruction manuals (instructions for use), and the like. The kit of the present invention is used for preparing a polyion complex (PIC) having a desired protein to be introduced into a target cell as a core part, and the prepared PIC can be effectively used as a protein delivery device to a target cell. Can be done.
[0044]
Examples
　The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
[0045]
1. Materials and Methods
1.1. Materials
α-Methoxy-ω-amino-poly (ethylene glycol) (MeO-PEG-NH 2 ; Mn = 12,000) was purchased from NOF (Tokyo, Japan). N-trifluoroacetyl-L-lysine N-carboxyanhydride (Lys (TFA) -NCA) was purchased from Chuo Kasei Seisakusho Co., Ltd. (Tokyo, Japan). Oxalyl chloride, 2-propion-3-methylmaleic anhydride, dichloromethane (CH 2 Cl 2 ), N, N-dimethylformamide (DMF), toluene, methanol and deuterium oxide (99.8 atomic% D) are from Tokyo Chemical Industry. Purchased from Co., Ltd. (Tokyo, Japan). Alexa Fluor 647 NHS Ester (Succinimidyl Ester) purchased from Thermo Fisher (Waltham, MA, USA) and DMSO-d 6 and Dulbecco's Modified Eagle's Medium (DMEM) purchased from Sigma Aldrich (St. Louis, MO, USA). However, bovine fetal serum (FBS) was purchased from Dainippon Sumitomo Pharmaceutical (Osaka, Japan). Cell Counting Kit-8 (CCK-8) was purchased from Dojin Chemical Research Institute (Kumamoto, Japan). The dialysis membrane was purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA) and the Vivaspin 6 centrifuge filter unit (including 10,000 MWCO (molecular weight cutoff), 30,000 MWCO and 100,000 MWCO) is Sartorius (Gottingen). , Germany).
[0046]
1.2. Instrument
　Proton Nuclear Magnetic Resonance ( 1 H-NMR) spectra are obtained using a JEOL ECS-400 spectrometer (JOEL Ltd., Japan) with a frequency of 400 MHz and the chemical shift is per million (1 H-NMR). Calculated as ppm). Polymer molecular weight distribution was measured by gel permeation chromatography (GPC) and organic phase GPC was performed on a TOSOH HLC-8220 system (Tosoh, Japan) with TSK gel G4000H HR and G3000H HR columns and poly (ethylene). The glycol) standard was used for calibration (Polymer Laboratories, Ltd., UK). Aqueous GPC measurements were performed on the JASCO LC-EXTREMA system (JASCO, Japan) equipped with the size exclusion column Superdex 200-10 / 300GL (GE Healthcare; USA). The size distribution and zeta potential were measured by Zetasizer Nano-ZS (Malvern, UK) through dynamic light scattering (DLS) and laser Doppler electrophoresis, respectively. Fluorescence intensity from the fluoresamine assay was measured via an ND-3300 nanodrop fluorescence spectrometer (Thermo Fisher, USA). UV / Vis spectroscopy was performed with a V-500 spectrophotometer (JASCO, Japan).
[0047]
1.3. Synthesis of PEG-poly (L-Lysine-CDM) Block Copolymers PEG-poly (L-
　lysine) block copolymers (PEG-p (Lys)) are obtained by slightly modifying the method described in [20] below. Prepared as.
MeO-PEG-NH 2 (Mn = 12,000) was reacted with Lys (TFA) -NCA to form PEG-p (Lys-TFA) by ring-opening polymerization, followed by deprotection of the trifluoroacetyl group. Briefly, MeO-PEG-NH 2 (1 g, 0.083 mmol) and Lys (TFA) -NCA (1.005 g, 3.75 mmol) were separately dissolved in 1 M thiourea containing DMF and then NCA solution. Was transferred to a PEG solution under an argon atmosphere and stirred at 35 ° C. for 3 days. The polymer was recovered as a white powder by precipitating in diethyl ether and drying under vacuum. The degree of polymerization was determined by 1 H-NMR method (DMSO-d 6 , 80 ° C), and the molecular weight distribution was examined by GPC (mobile phase: 10 mM LiCl containing DMF; temperature: 40 ° C; flow velocity: 0.8 mL / min; Detector: Refractive index). In addition, the protecting group TFA was removed by treatment with 1 M NaOH in methanol solution overnight at 35 ° C. followed by dialysis against water using a 6-8 kD MWCO dialysis membrane. After lyophilization, the final product was obtained as a white powder. The component of the deprotected polymer is 1Analysis was performed by 1 H-NMR method (D 2 O, 25 ° C). In the 1 H-NMR spectrum, the composition of the PEG-p (Lys) block copolymer was determined by the intensity ratio of the peaks derived from the protons of -OC H 2 C H 2 of PEG and -C 3 H 6 of lysine . The molecular weight distribution was analyzed by GPC (mobile phase: 10 mM acetate and 500 mM NaCl acetate-buffered saline (pH 3.3); room temperature; flow velocity: 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, a CDM acyl chloride (CDM-Cl) was prepared with minor modifications to the description in the previous report [21] . 2-Propion-3-methylmaleic anhydride (CDM, 200 mg, 1.09 mmol) was dissolved in anhydrous toluene and evaporated under vacuum. CDM was dissolved in anhydrous CH 2 Cl 2 (15 mL), followed by addition of oxalyl chloride (4 mL, 5.9 g, 46 mmol) and reaction with CDM at room temperature for 12 hours. Next, CH 2 Cl 2 and residual oxalyl chloride were removed by evaporation to give a clear oil. Subsequently, CH 2 Cl 2 (4 ml) was added to dissolve CDM-Cl, while CH 2 Cl 2 (20 ml) was used to dissolve PEG-p (Lys) (200 mg, 0.011 mmol). The PEG-p (Lys) solution was then transferred to the CDM-Cl solution and the reaction was stirred at room temperature. After 12 hours, the product was recovered by diethyl ether precipitation and overnight vacuum drying. The final product was analyzed by 1 H-NMR and GPC.
[0049]
S1. Chemical reaction scheme, polymer synthesis and chemical analysis
[Chemical 18]

[0050]
[Changing 19]

[0051]
[Changing 20]

[0052]
1.4. Preparation of protein-free core cross-linked polyion complex (PIC) micelles (empty PIC micelles), and stable
　polymer solutions (1 mg / mL) at various pH conditions, pH 4 or 5 acetate buffer, or Prepared in phosphate buffer (10 mM acetic acid or phosphate with 150 mM NaCl) at pH 6.5 or 7.4. The polymer was dissolved in buffer at different pH (1 minute vortex, 1 hour incubation). The solution was filtered through a 0.22 μm syringe filter followed by DLS measurements. In addition, a polymer solution was prepared in deuterated phosphate buffer (10 mM) at pH 7.4, and analyzed by 1 H-NMR method before and after the addition of deuterated hydrochloric acid (DCl).
[0053]
　In addition, empty PIC micelles autonomously associated with pH 7.4 buffer were allowed to stand in 10 mM phosphate buffer at pH 6.5 or 7.4 with 150 mM NaCl with a final concentration of 0.5 mg / ml of polymer. The stability of empty PIC micelles under the conditions was evaluated over time by DLS. The 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 fetal kidney cell line 293 (HEK 293) cell line. In this experiment, PEG-p (Lys) was used as a control. Cells were seeded 3000 cells per well on 96-well plates in DMEM medium with 10% FBS and incubated at 5% CO 2 , 37 ° C. for 24 hours. The cells were then exposed to each concentration of polymer. After 48 hours of incubation with the polymer, cytotoxicity was assessed by the CCK-8 assay by measuring formazan absorbance at 450 nm. In addition, the PEG-p (Lys-CDM) block copolymer was dissolved in DMEM (1 minute vortex, 1 hour incubation) and the resulting solution was evaluated by DLS.
[0055]
1.6. Preparation and physicochemical evaluation of myoglobin-encapsulating micelles (myo / m)
　Empty PIC micelles prepared by dissolving PEG-p (Lys-CDM) polymer (3 mg / mL) in pH 5 buffer (10 mM acetate). Formation was suppressed and a 0.1 molar equivalent of 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. The micelles were then purified and bound by ultrafiltration using a 100,000 MWCO centrifuge filter using phosphate buffered saline (10 mM phosphate containing 150 mM NaCl) at pH 7.4. No proteins and polymers were removed. In addition, to assess encapsulation efficiency, myoglobin was labeled with Alexa Fluor 647 succinimidyl ester and the mixed solution was GPC (10 mM phosphate buffer containing 150 mM NaCl with pH 7.4 as mobile phase; flow rate 0.75 mL / Analyzed by minutes; room temperature).
[0056]
An excitation wavelength of 650 nm and an emission wavelength of 668 nm were used for the fluorescence detection settings. Encapsulation efficiency was calculated by dividing the amount of encapsulated protein by the amount of protein added. In addition, fluorescence correlation spectroscopy (FCS) was used to quantify the amount of Alexa Fluor 647-labeled myoglobin contained per micelle. FCS experiments were performed at room temperature using an MF-20 (Olympus, Japan) equipped with a laser beam with a wavelength of 633 nm. In addition, lysozyme and albumin were also encapsulated in micelles by the same method and the size of micelles was determined by DLS.
[0057]
1.7. Preparation and physicochemical evaluation of CDM-modified myoglobin-encapsulated micelles (CC-myo / m) CDM-modified myoglobin (CC-myo) -encapsulated micelles (CC-myo / m) are few in the method
reported [14,16] . It was prepared as a control micelle according to the modified method. Briefly, myoglobin was dissolved in 0.1 M NaHCO 3 buffer to make a 2 mg / mL solution and stirred at 4 ° C for 30 minutes. Then 50 mol equivalents of CDM was slowly added to the solution and stirred at 4 ° C. for 2 hours. This myoglobin solution was purified by ultrafiltration using a 10,000 MWCO centrifugal filter. The efficiency of CDM modification was determined by the fluorescamine method using the Nanodrop Fluorescence Spectrometer (Thermo Fisher, USA) and the percentage of converted amines was calculated according to the previously reported method [16] . Subsequently, CC-myo / m was prepared by mixing PEG-p (Lys) with charge-converted myoglobin, and a phosphate buffered physiology with a pH of 7.4 with a 2: 1 N / C (amino / carboxyl group) ratio. Titrated in saline. In addition, a mixture of PEG-p (Lys) and natural myoglobin was used as a control at the same polymer-to-protein molar equivalent. The size distribution, polydispersity index (PDI) and zeta potential of micelles were analyzed by Zetasizer Nano ZS.
[0058]
1.8. Stability of myoglobin-encapsulated micelles to different salt concentrations and different pH buffers
　Dilute the sample to 0.5 mg / m to test the in vitro stability of myo / m and CC-myo / m to different pH conditions. The polymer concentration was mL. Micelle was incubated in 10 mM phosphate buffer at pH 6.5 or pH 7.4 with 150 mM NaCl solution and measured by DLS over time (25 ° C). The size distribution, PDI and obtained count rate were recorded on the Zetasizer Nano ZS. Furthermore, the stability of micelles was investigated by shielding the electrostatic interaction with a high-concentration salt buffer solution. Myo / m and CC-myo / m were prepared and diluted to a polymer concentration of 0.5 mg / mL. Micelle solutions were dialyzed in 5 L 10 mM phosphate buffer containing 600 mM NaCl at pH 7.4 and pH 6.5 in a 20,000 MWCO dialysis cassette. At different time points, samples were taken from inside the dialysis cassette and analyzed with DLS to follow the disintegration of micelles.
[0059]
1.9. Myoglobin release from myo / m at different pH conditions
　Alexa Fluor 647 labeled myo / m in 5 L 10 mM phosphate buffer and 150 mM NaCl, using a dialysis cassette with 20,000 Da MWCO, Dialysis was performed at pH 7.4 and pH 6.5 at room temperature. Samples were taken from the inside of the dialysis cassette at a given time point and the fluorescence intensity was evaluated using a NanoDrop 3300 fluorescence spectrometer.
[0060]
1.10. Assessment of Myoglobin Activity
　Myoglobin is released from micelles by incubation overnight at pH 6.5 under dilution conditions of 10 mM phosphate buffer + 150 mM NaCl, followed by ultrafiltration with a centrifugal filter of 30,000 MWCO. I let you. Filter passages were collected and then concentrated to 0.05 mg / mL by ultrafiltration using a 10,000 MWCO centrifugal filter. Myoglobin activity was assessed based on previously reported methods [22] . 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 reduced by adding 5 equivalents of an aqueous solution of sodium dithionite (NaS 2 O 4 ). Subsequently, the reduced myoglobin was oxygenated by introducing O 2 for 30 minutes and then reduced by bubbling argon for 2 hours. The oxygenation / reduction cycle was repeated many times according to the previously reported protocol [22] . As a control, the same concentration of natural myoglobin was used.
[0061]
1.11. In vivo Blood retention and biodistribution
　Using Alexa Fluor 647 labeled myoglobin, myo / m, CC-myo / m and free myoglobin were prepared and Nikon A1R confocal laser scanning microscope (IV- CLSM) (Nikon, Japan) was used to track the blood retention and biodistribution of myoglobin. Five-week-old Balb / c female mice were injected with 100 μL of a sample solution containing 100 μg / mL fluorescently labeled myoglobin under anesthesia by tail vein administration, and the ear capillaries were observed [23] . Fluorescence intensity in the earlobe vein and skin was continuously measured. Twelve hours after injection, mice were euthanized, organs (kidney, liver and spleen) were harvested and imaged ex vivo by IV-CLSM. Thirty minutes prior to euthanasia and organ collection, 100 μL of Hoechst 33342 solution was administered via the tail vein for nuclear staining. In addition, Alexa Fluor 647 labeled and unlabeled myoglobin were used to prepare myo / m and CC-myo / m to track the retention of the polymer in the micelles in the blood. Mice were administered a 100 μL sample solution with a 2 mg / mL fluorescently labeled polymer through the tail vein and the ear capillaries were imaged under a microscope. All animal experiments in this test were conducted in accordance with the University of Tokyo Experimental Animal Handling Regulations.
[0062]
1.12. Protein and Polymer Labeling Protein labeling
with Alexa Fluor 647 succinimidyl ester was carried out with a slight modification to the manufacturer-supplied method. Briefly, 5 mg / ml protein was dissolved in 0.15 M sodium bicarbonate buffer, while 0.5 mol equivalent of Alexa Fluor 647 succinimidyl ester was dissolved in DMF to make a 10 mg / ml solution. The above two solutions were mixed and reacted at room temperature for 1 hour. The solution was then run on a Sephadex G-25 column for purification by gel permeation chromatography. After purification, Alexa Fluor 647 labeled protein was lyophilized for further use. Labeling and purification of PEG-p (Lys) was performed in the same manner as for proteins. However, since PEG-p (Lys-CDM) has self-assembling properties, labeling should be performed in 10 mM phosphate buffer (pH 6.5), gel filtration should be performed to remove free dye, and then a polymer solution. Was treated with 0.1 N HCl for 5 minutes and immediately lyophilized.
[0063]
1.13. Fluorescence Correlation Microscope
　Fluorescence Correlation Spectroscopy (FCS) experiments were performed at room temperature on an MF-20 (Olympus, Japan) equipped with a laser beam with a wavelength of 633 nm. Alexa Fluor 647-labeled myoglobin and Alexa Fluor 647-labeled myoglobin-encapsulated micelle solutions were placed in pretreated 384-well glass-bottomed microplates at 30 μL / well. A standard 633 nm solution (Olympus, Japan) with a molecular weight of 652 Da was also placed on the plate to determine the structural parameters. Samples were excited with a 633 nm laser beam and scanned 5 times for 20 seconds each. The obtained data was fitted by the automatic fitting function of the software.
[0064]
2. Results and discussion
2.1. Synthesis and chemical analysis of block copolymers Lys (TFA) polymers are initiated by the terminal primary amino group of
　MeO-PEG-NH 2 [20] . ) -Synthesized by ring-opening polymerization of NCA. As a result of GPC analysis of the polymerized polymer, a narrow molecular weight distribution (M w / M n = 1.03) was shown (Fig. 11). After removing the protecting group TFA by alkaline hydrolysis, the proton ratio of -OCH 2 CH 2- (δ = 3.5 ppm) of PEG and -C 3 H 6 (δ = 1.2 ppm to 1.8 ppm) of p (Lys) When the degree of polymerization (DP) was confirmed by 1 H-NMR used , the DP of lysine was 37. Furthermore, as a result of GPC (pH 3.3 acetate-buffered saline of 10 mM acetate containing 500 mM NaCl as mobile phase; flow velocity 0.75 mL / min), a monomodal peak showing a narrow molecular weight distribution was shown (Fig. 12). ).
[0065]
Next, CDM was introduced into the polymer by reacting CDM-Cl with a primary amine of PEG-p (Lys). Then, by comparing the peak intensity of -CH 3 (δ = 2.0 ppm) on CDM with the methylene peak on PEG and the β, γ and δ-methylene protons of lysine, the introduction amount and introduction rate of CDM can be determined. confirmed. The CDM unit was calculated to be about 17, and the CDM addition rate was about 45%. In addition, PEG-p (Lys-CDM) showed a narrow molecular weight distribution by GPC using an acetate buffer solution with pH 3.3 (10 mM acetic acid with 500 mM NaCl) as the mobile phase (Fig. 13). These results indicate that PEG-p (Lys-CDM) could be synthesized with the quality required for micelle preparation.
[0066]
2.2. Preparation of protein-free core cross-linked polyion complex (PIC) micelles (empty PIC micelles) and stability at various pH conditions
　PEG-p (Lys-CDM) is an amine moiety and an amine-reactive CDM unit. Having both, it may be in the form of a free polymer under acidic pH environment due to the protonation of amines and the formation of CDM rings. On the other hand, at near neutral pH, the CDM group forms a stable amide bond with the amine, forming a carboxyl group for the formation of a further polyion complex (Scheme S2). Therefore, we evaluated the structure of PEG-p (Lys-CDM) by DLS after incubating the polymer at different pH for 1 hour.
[0067]
PEG-p (Lys-CDM) was found to autonomously associate with micelles at pH 7.4 (which is higher than other pH values). The derived count rate is determined by DLS and correlates with the presence of large or dense particles [24] (Fig. 2a). The resulting micelles showed a size of about 40 nm and a PDI of 0.2 at pH 7.4. On the other hand, at pH below 6.5, the count rate remained low, indicating that PEG-p (Lys-CDM) did not associate with micelles. Elimination of proton peaks from polyamino acids and side chain structures of PEG-p (Lys-CDM) by measuring 1 H-NMR of the polymer in deuterated phosphate buffer (10 mM) at pH 7.4 Heading, this indicates a decrease in the mobility of the polyamino acid backbone due to the binding of the amine to the CDM moiety (Fig. 14a). After adding 2 M heavy hydrochloric acid to the above solution, peaks from polyamino acids and side chain structures recovered to 75% after 10 minutes of incubation (Fig. 14b). This indicates the release of polyamino acids under low pH conditions. The pH-dependent micelle formation of PEG-p (Lys-CDM) should be noted to avoid the formation of empty micelles before adding the protein.
[0068]
　The stability of empty PIC micelles autonomously associated at pH 7.4 was evaluated by DLS after diluting the micelles to solutions showing different pH. At pH 7.4, the size of empty PIC micelles went from 43 nm to 38 nm in 24 hours (Figure 3), PDI variability was small, and the count rate was only attenuated by 20%. On the other hand, at pH 6.5, empty PIC micelles were unstable, showing a rapid decrease in size and count rate, as well as an increase in PDI above 0.4 in the first 5 hours of incubation (Figure 3). At pH 6.5, measurement of micelle size after 5 hours was unreliable due to the high PDI and was omitted. These results indicate that empty PIC micelles are pH responsive and decay.
[0069]

　2.3. Determining whether PEG-p (Lys-CDM) can be safely used as a delivery carrier for the biological application of PEG-p (Lys-CDM) in vitro cytotoxic protein-encapsulating micelles to HEK293 cells. is important. Therefore, the cytotoxicity of PEG-p (Lys-CDM) was investigated by culturing PEG-p (Lys-CDM) together with HEK 293 cells for 48 hours. The PEG-p (Lys) polymer was used as a control because it is a precursor of PEG-p (Lys-CDM) and is widely used as a delivery carrier.
[0070]
　As shown in Figure 4, PEG- (Lys-CDM) is less cytotoxic at all polymer concentrations than PEG-p (Lys), with a polymer concentration of 1 mg / mL greater than 70%. Cell viability was maintained. The low toxicity of PEG- (Lys-CDM) is believed to be due to autonomous association to empty PIC micelles under medium conditions, as indicated by DLS assessment of PEG- (Lys-CDM) in DMEM (Fig. 15). Be done. These results indicate that PEG-p (Lys-CDM) is a highly safe delivery carrier.
[0071]
2.4. Preparation of protein-encapsulated micelles by precise control of pH
　Proteins are composed of a large number of negatively charged groups (glutamate, aspartic acid, and C-terminal carboxyl groups) and positively charged groups (lysine, arginine, and N-terminal amines). ) Is a macromolecule with a non-uniformly charged surface. Thus, PEG-p (Lys-CDM) can form a PIC with a carboxyl group in the protein and covalently attach to a primary amino group in the protein via a pH-responsive CDM moiety (Scheme S3). In addition, amines in PEG-p (Lys-CDM) can further crosslink the micellar core by reacting with the CDM group that did not bind to the protein.
[0072]
　As observed above (Fig. 2), PEG-p (Lys-CDM) can autonomously associate with micelles at the pH of the medium. Since PEG-p (Lys-CDM) exists as a free polymer at pH 5, PEG-p (Lys-CDM) is dissolved in 10 mM acetate buffer (pH 5) to prepare a polymer solution, which is empty. Suppressed PIC micelle formation. In addition, a protein solution was prepared in 10 mM phosphate buffer (pH 8) and mixed with the polymer solution to form a polyion complex with lysine residues in PEG-p (Lys-CDM), and the CDM moiety. Self-assembly was performed through amide formation with. After mixing the polymer solution and the protein solution, the pH was adjusted to 7.4. Since free proteins and micelles show different elution times in GPC, myoglobin inclusion efficiency was determined by GPC. Myoglobin was fluorescently labeled with Alexa Fluor 647 for fluorescence detection. Encapsulation efficiency was calculated by dividing the amount of encapsulated protein by the amount of protein added.
[0073]
　As shown in Table 1, myoglobin (17.6 kDa protein with an isoelectric point 7) was encapsulated in micelles with an efficiency of 62% and 5% by weight, resulting in micelles with a PDI of 0.18 and a size of 40 nm. After purifying the micelles by ultrafiltration with phosphate buffered saline (pH 7.4, 10 mM phosphate buffer with 150 mM NaCl), the number of myoglobins contained per micell using FCS. Was quantified. By calculating the ratio of count rates per molecule between micelles and Alexa Fluor 647-labeled myoglobin, we confirmed that approximately two Alexa Fluor 647-labeled myoglobins were included per micelle (Table 2).
[0074]
[table 1]

[0075]
[Table 2]

[0076]
　In addition to myoglobin, bovine serum albumin (BSA) and lysozyme were also selected to evaluate the inclusion capacity of micelles because they have a different size (molecular weight) and net charge (isoelectric point) than myoglobin. As a result, it was shown that PEG-p (Lys-CDM) can encapsulate these proteins in micelles (Table 3). Furthermore, TEM observations revealed the particle morphology of these protein-encapsulating micelles (Fig. 16). These results indicate that the micelle system of the present invention for encapsulating proteins has versatility.
[0077]
S4. Inclusion of different proteins in polymer micelles
As shown in Table 3, PEG-p (Lys-CDM) uses various proteins with different molecular weights and isoelectric points (pI) in a narrow particle size distribution. I was able to form micelles.
[0078]
[Table 3]

[0079]
2.5. Preparation of control myoglobin-encapsulated micelles
　To evaluate the effectiveness of myo / m prepared in the previous section, control micelles without covalent bonds were constructed. In order to prepare control micelles, we first modified CDM to myoglobin by gradually adding CDM to myoglobin solution. The CDM introduction rate was 92.8% compared to the fluorescamine method, and the zeta potential of CC-myo was -29.5 mV, which was lower than the zeta potential of natural myoglobin (-9.2 mV). This indicates that the charge was converted by the introduction of CDM. Subsequently, in phosphate buffered saline (10 mM phosphate buffer containing 150 mM NaCl, pH 7.4), PEG-p (Lys) was added at an N / C (amino / carboxyl group) ratio of 2: 1. PIC micelles were prepared by mixing with CC-myo, and a mixture of PEG-p (Lys) and natural myoglobin as a control was prepared at the same N / C ratio as above. CC-myo formed PIC micelles with PEG-p (Lys) via electrostatic interaction (Table 1). However, myoglobin without CDM modification did not form micelles with PEG-p (Lys). It is considered that this is because the non-uniform surface charge of myoglobin is disadvantageous to the stable polyion complex [4] .
[0080] [0080]
2.6. Micelle stability
　Micelle stability was investigated using buffers with different salt concentrations and pH.
　First, the pH stability test was conducted on micelles (myo / m) consisting of PEG-p (Lys-CDM) and control micelles (CC-myo / m) in 10 mM phosphate buffered saline (pH 6.5 or pH 7.4). ) Was evaluated. Micelle size and PDI were measured hourly by DLS. It was shown that myo / m had high stability with no change in size and PDI at both pH 7.4 and 6.5. CC-myo / m showed high stability at pH 7.4, as shown in Figure 5. On the other hand, at pH 6.5, CC-myo / m rapidly destabilized. In addition, myo / m was salt-tolerant at both pH 6.5 and pH 7.4 (Fig. 5), while empty PIC micelles disintegrated rapidly under the same conditions (Fig. 3). This suggests that the protein stabilizes micelles of PEG-p (Lys-CDM).
[0081]
The biological application of PIC micelles is difficult because the 　electrostatic interaction that retains the micelle structure dissociates during blood retention [25,26] . Therefore, high NaCl concentration (600 mM) completely inhibits electrostatic interactions [25,27] in micelles, so for 5 L of 10 mM phosphate buffer containing 600 mM NaCl at pH 7.4 or 6.5. The stability of micelles was evaluated by dialysis using a 20,000 MWCO dialysis cassette under diluting conditions. Samples were taken over time and DLS analysis was performed to track micellar stability. The control CC-myo / m based solely on PIC dissociated immediately after standing under high salinity, while myo / m had a size and count rate after 24 hours at pH 6.5 compared to pH 7.4. It dropped rapidly. This indicates that rapid degradation of micelles occurs at acidic pathophysiological pH, while it has strong stability at physiological pH (Fig. 6).
[0082]
2.7. Myoglobin release
　from myo / m The release of myo / m from micelles is dialed with Alexa Fluor 647 labeled myo / m encapsulated micelles against 5 L of 10 mM phosphate buffered saline at pH 7.4 and pH 6.5. It was evaluated by. Here, the fluorescence intensity of the micelles in the dialysis cassette was measured over time. At pH 7.4, myo / m released the contained protein slowly (Fig. 7). On the other hand, the release of myoglobin from micelles was promoted at pH 6.5, and within 24 hours, about 70% of the encapsulated protein was released (Fig. 7). These results correlate with the stability of micelles at pH 7.4 and rapid degradation at pH 6.5, strongly suggesting that micelles respond to pathological pH and ionic strength (150 mM NaCl). ..
[0083]
2.8. Myoglobin activity Oxygenation of myoglobin can be determined by the movement of
　the Soret band (380-460 nm) and the Q band (480-650 nm) [22,28-30] . Therefore, the activity of myoglobin released from myo / m at pH 6.5 was evaluated by UV / Vis spectroscopy. Addition of sodium dithionite to the released myoglobin solution revealed a Soret band at 434 nm. This corresponds to the band of reduced myoglobin. In addition, a blue shift in the Soret band from 434 nm to 414 nm, as well as a peak split in the Q band, was observed after the introduction of O 2 . This corresponds to the band of oxygenated myoglobin [22,28,29] .
[0084]
After that, when Ar gas was bubbled, the reverse change of Soret band and Q band appeared, and deoxygenation was confirmed (Fig. 8a). In addition, the released myoglobin succeeded in structurally changing to oxygenated and reduced myoglobin by alternately bubbling O 2 or argon gas (Fig. 8c). As a control, natural myoglobin was used (Fig. 8b, d). Moreover, there were no significant differences in oxygenation or deoxygenation between natural myoglobin or myoglobin released from myo / m. These results indicate that the proteins contained in myo / m retain their functionality upon release.
[0085]
2.9. In vivo blood retention and biodistribution
　Many therapeutic proteins have reduced blood retention due to blood aggregation and rapid renal excretion [31,32] . In this example, it is known that PEG-p (Lys-CDM) -based micelles aggregate in blood and cause renal excretion as a model protein for testing the ability of proteins to improve their pharmacokinetics [34]. I used myoglobin. Myoglobin was fluorescently labeled with Alexa Fluor 647 and encapsulated in micelles to examine in vivo blood retention and biodistribution.
[0086]
Fluorescently labeled myo / m showed a similar size distribution for unlabeled micelles. After intravenous injection, the retention of fluorescently labeled micelles in the blood was recorded by real-time IV-CLSM. As shown in Figures 9a-c, covalently stabilized myo / m showed a half-life of more than 120 minutes, while CC-myo / m (10 minutes) and free myoglobin (9 minutes) were short. Showed half-life. In addition, CC-myo / m and free myoglobin showed strong fluorescent signals in the skin real tissue, but myo / m did not escape to the skin. This indicates that the encapsulated myoglobin did not leak from the micelles in the blood.
[0087]
Next, myo / m and CC-myo / m prepared from Alexa Fluor 647 labeled polymer and unlabeled myoglobin were used to evaluate in vivo blood retention and biodistribution of the polymer. Myo / m showed a half-life of 120 minutes or more, similar to that labeled with myoglobin, but CC-myo / m had a half-life of only 1 minute and was not detected in blood after 5 minutes. Since PEG-p (Lys) is rapidly excreted from the blood within a few minutes, CC-myo / m is considered to be unstable in the blood. This corresponds to the fact that the half-life of fluorescently labeled myoglobin in CC-myo is similar to the half-life of myoglobin alone (Fig. 9a, b), and charge-converted myoglobin micelles rapidly in the blood. It shows that it is disassembled. On the other hand, myo / m shows high stability in blood (Fig. 9c, e). This is because the retention in blood of the fluorescently labeled polymer PEG-p (Lys-CDM) corresponds to the retention in blood of the fluorescently labeled protein.
[0088]
　The biodistribution of myoglobin, CC-myo / m and myo / m was evaluated 12 hours after dosing in the major organs involved in nanoparticle excretion, namely kidney, liver and spleen.
　Cell nuclei were stained by administration of Hoechst to the tail vein 30 minutes prior to imaging. The kidneys, liver and spleen were then harvested and observed by ex vivo fluorescence imaging. As shown in FIGS. 10a-c, free myoglobin and CC-myoglobin show high accumulation in the kidney, consistent with the rapid excretion of free myoglobin and CC-myo / m from the blood. On the other hand, myo / m micelles suppressed the accumulation in the kidney as compared with CC-myo / m and myoglobin, and accumulated in the liver.
[0089]
　In addition, for CC-myo / m tracked with Alexa Fluor 647-labeled PEG-p (Lys), rapid excretion of the polymer resulted in little fluorescent signal being detected in the kidney, liver and spleen (Fig. 10d). .. On the other hand, the signal from myo / m tracked using Alexa Fluor 647 labeled PEG-p (Lys-CDM) was observed mainly in the liver (Fig. 10e), which is the fluorescently labeled myoglobin-encapsulating myo / m. Consistent with the distribution (Fig. 10c). These results demonstrate the high stability of myo / m in blood, indicating that PEG-p (Lys-CDM) is useful for preparing protein-encapsulating micelles for in vivo delivery. show.
[0090]
3. Conclusion
　The present inventor uses a novel high molecular weight PEG-p (Lys-CDM) capable of encapsulating a protein by a combination of a polyion complex and a pH-responsive amide bond, and has a high pH-responsiveness for protein encapsulation. Succeeded in developing molecular micelles. Using myo / m as a model, we have shown that these micelles are stable at pH 7.4 but rapidly disintegrate at pH 6.5. Furthermore, the micelles of the present invention containing myoglobin showed excellent blood retention in vivo as compared with micelles assembled alone by free myoglobin and PIC formation. Furthermore, myoglobin released from micelles at pH 6.5 was shown to have the same oxygenation and reducing ability as natural myoglobin, indicating that the micelles of the present invention can retain protein function. These findings indicate the potential of the micelles of the present invention as protein nanocarriers that target pathological tissues and are effective in controlling protein activity spatiotemporally in vivo.
[0091]
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[0093]
Example 2
1. Preparation of IL-12-encapsulated micelle In
this example, IL-12-encapsulated micelle was prepared by precisely controlling the pH. Briefly, 2.5 mg of PEG-P (Lys-CDM) is dissolved in 0.5 mL of 20 mM phosphate buffer (pH 5) to prevent the polymer from autonomously associating to form empty micelles. It was allowed to stand for 1 hour. 10 μg of IL-12 was dissolved in 0.5 mL of 20 mM phosphate buffer (pH 8). The IL-12 solution was added to the polymer solution at a rate of 5 μL / min under stirring (shaking), followed by continuous stirring (shaking) for 6 hours. 1 mL of buffer (pH 8) was then added to the mixture and the mixture was stirred (shaken) overnight.
[0094]
　Encapsulation efficiency was measured by the ELISA method. The concentration of unencapsulated free IL-12 in the mixture was detected by an ELISA kit and the amount of encapsulated IL-12 was calculated.
　As a result, the concentration of free IL-12 in the 2 mL mixture was 1.6 μg / mL. Since the total concentration of IL-12 was 5 μg / mL, the inclusion efficiency was 68%.
[0095]
2. Purification and characterization of IL-12-encapsulated micelles
　Purification was performed by dialysis. The mixed solution was loaded into a dialysis cassette containing 100 kDa MWCO and dialyzed against 10 mM phosphate buffer (pH 7.4) and 150 mM NaCl overnight at 4 ° C. The purified micelle solution was then precisely adjusted in concentration (adjusted to have a polymer concentration of 1 mg / mL) for size and zeta potential measurements with the Zetasizer.
　As a result, the average z size by DLS was 43 nm and the PDI was 0.229 (Fig. 17). The surface of the micelle was slightly negatively charged and the zeta potential was -4.1 ± 1.0 mV.
[0096]
3. In vitro drug release experiment In
　this section, the dialysis method was used again. The purified micelle solution was loaded into a dialysis cassette with 100 kDa MWCO, 500 mL of 10 mM phosphate buffer (pH 7.4) + 150 mM NaCl, and 500 mL of 10 mM phosphate buffer (pH 6.5) +. 150 mM NaCl was dialyzed separately at room temperature. At a given point in time, the solution was taken 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 under pH 6.5 was about four times the amount released under pH 7.4 (Fig. 18).
[0097]
4. In vitro cell experiment In
　this section, the amount of INF-γ secretion from mouse splenocytes was measured in order to evaluate the bioactivity of micelles and released IL-12.
　9-week-old BALB mice were sacrificed and spleen cells were collected from the spleen. The collected splenocytes were then seeded on 96-well plates at a concentration of 1 × 10 5 cells per well. IL-12 released from micelles was isolated by dialyzing the micelle solution against buffer (pH 5) and then ultracentrifuging the external solution to adjust the concentration. Micelle and released IL-12 were added to the wells at different concentrations and natural IL-12 was used as standard. After standing for 24 hours and 48 hours, the supernatant in each well was removed, and the INF-γ concentration was measured by an ELISA kit.
[0098]
　As a result, after 24 hours, the increase in INF-γ concentration was significantly suppressed compared to IL-12 released in IL-12-encapsulated micelles, and thus the binding of IL-12 to the receptor was suppressed by micelle formation. It shows that it was done (Fig. 19). The difference between released IL-12 and native IL-12 is not significant, indicating that micellization does not affect the bioactivity of the encapsulated protein. After 48 hours, the difference between the three groups decreased. This phenomenon is due to the collapse of micelles.
The scope of the claims
[Claim 1]
　A polymer complex containing a protein and a block copolymer represented by the following formula (1).
[Chemical formula 21]

[In the formula, R 1 and R 2 are each independently a hydrogen atom, or a linear or branched alkyl group having 1 to 12 carbon atoms which may be substituted, or an azide. Representing an amine, maleimide, ligand or labeling agent,
　R 3 represents a compound represented by the following formula (I),
[Chemical formula 22]

(in the formula, R a and R b are independently hydrogen atoms, respectively. Alternatively, it represents an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group which may be substituted. A and R b may be bonded to each other to form an aromatic ring or a cycloalkyl ring together with the carbon atom to which each is bonded . The bond between the carbon atoms to which R a and R b are bonded, respectively, may be formed. , It may be a single bond or a double bond.)
　L 1Is NH, CO, or the following equation (11) :-(
　　CH 2 ) p1 -NH- (11)
(in the equation, p1 represents an integer from 1 to 6)
, or the following equation (12 ). ):
　　-L 2a- (CH 2 ) q1 -L 3a- (12)
(In the equation, L 2a stands for OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a stands for NH or CO. Represents. Q1 represents an integer from 1 to 6)
, and
　m1 and m2 independently represent an integer from 0 to 500 (provided that the sum of m1 and m2 is an integer from 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.
　The notation of "/" indicates that the sequence order of each of the (m1 + m2) monomer units shown on the left and right of the "/" is arbitrary. ]
[Claim 2]
　The complex according to claim 1, wherein the compound represented by the formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig).
[Chemical 23]

[Claim 3]
　The complex according to claim 2, wherein the compound represented by the formula (I) is a compound represented by the following formula (Ia) or (Ib).
[Chemical 24]

[Claim 4]
　The complex according to claim 1, wherein the block copolymer represented by the formula 1 is represented by the following formula (2).
[Chemical 25]

[Claim 5]
　The complex according to claim 1, wherein the protein is covalently bonded to the block copolymer represented by the formula 1.
[Claim 6]
　The complex according to claim 5, wherein the covalent bond is cleaved in a pH-dependent manner.
[Claim 7]
　A protein delivery device selected from cell surface, intracellular and extracellular, comprising the polymer complex according to any one of claims 1 to 6.
[Claim 8]
　A protein delivery kit to any of cell surface, intracellular and extracellularly selected, comprising the block copolymer represented by the following formula (1).
[Chemical formula 26]

[In the formula, R 1 and R 2 are each independently a hydrogen atom, or a linear or branched alkyl group having 1 to 12 carbon atoms which may be substituted, or an azide. Representing an amine, maleimide, ligand or labeling agent,
　R 3 represents a compound represented by the following formula (I), and
[Chemical Formula 27]

(in the formula, R a and R b are independently hydrogen atoms, respectively. Alternatively, it represents an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group which may be substituted. A and R b may be bonded to each other to form an aromatic ring or a cycloalkyl ring together with the carbon atom to which each is bonded . The bond between the carbon atoms to which R a and R b are bonded, respectively, may be formed. , It may be a single bond or a double bond.)
　L 1 is NH, CO, or the following equation (11) :-(
　　CH 2 ) p1 -NH- (11)
(in the equation, p1 represents an integer from 1 to 6)
, or the following equation ( 12):
　　-L 2a- (CH 2 ) q1 -L 3a- (12)
(In the equation, L 2a stands for OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, and L 3a stands for NH or CO. Represents a group represented by (q1 represents an integer of 1 to 6)
, and
　m1 and m2 independently represent an integer of 0 to 500 (however, the sum of m1 and m2 represents an integer of 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.
　The notation of "/" indicates that the sequence order of each of the (m1 + m2) monomer units shown on the left and right of the "/" is arbitrary. ]
[Claim 9]
　The kit according to claim 8, wherein the compound represented by the formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig).
[Chemical 28]

[Claim 10]
　The kit according to claim 9, wherein the compound represented by the formula (I) is a compound represented by the following formula (Ia) or (Ib).
[Chemical 29]

[Claim 11]
　The kit according to claim 8, wherein the block copolymer represented by the formula 1 is represented by the following formula (2).
[Changing 30]