Multi-stimulus responsive pigment epithelium-derived factor-elastin multi-block copolypeptide for Anti-angiogenesis, self-assembled multivalent nanostructure, and pharmaceutical composition thereof

Abstract

An embodiment of the present application provides a stimulus-responsive multi-block copolypeptide comprising a peptide derived from pigment epithelium-derived factor (PEDF), a drug carrier, and a pharmaceutical composition thereof. The drug carrier can exhibit improved antiangiogenic activity by forming a multivalent nanostructure of which the size has been controlled through His-Zn2+ chelation.

Description

Antiangiogenic multi-stimulus responsive pigment epithelium-derived factor-elastin multi-block copolypeptide, multivalent self-assembled nanostructure, and pharmaceutical composition thereof

[0001] The present application relates to a multi-stimulus responsive pigment epithelium-derived factor-elastin multi-block copolypeptide for anti-angiogenic purposes, a multivalent self-assembling nanostructure, and a pharmaceutical composition thereof.

[0002] Angiogenesis is the biological process that provides new blood vessels to tissues and organs. Specifically, it refers to the formation of new capillaries from pre-existing microvessels. It is a fundamental process of blood vessel formation in the body after growth. Normally observed physiological angiogenesis in the human body occurs only in very limited circumstances, such as embryonic and fetal development, uterine maturation, placental proliferation, corpus luteum formation, and wound healing. Even during these periods, angiogenesis is tightly regulated, and it ceases once the required function is achieved. New angiogenesis is tightly regulated by angiogenic regulators, and its phenotype has been reported to be altered by the overall balance between the up-regulation of angiogenic stimulators and the down-regulation of angiogenic inhibitors.

[0003] Recently, research into the development of angiogenesis inhibitors has been actively conducted, as animal cancer models and human clinical trials have demonstrated that inhibiting tumor angiogenesis can effectively inhibit tumor growth and development and prolong patient life.

[0004] These angiogenesis inhibitors are particularly in the spotlight in anticancer treatment for the following reasons: first, angiogenesis inhibitors have the potential to be commonly used in all solid tumors; second, existing anticancer chemotherapy uses the rapid growth of cancer cells to treat them, so it shows toxic effects on bone marrow cells and gastrointestinal cells with relatively fast cell cycles, whereas angiogenesis inhibitors have the advantage of relatively few side effects even with long-term administration; third, because a single blood vessel cell supplies nutrients and oxygen to hundreds of cancer cells, many cancer cells can be inhibited by inhibiting a single blood vessel cell; and fourth, while existing anticancer therapies cause anticancer drugs to leak out of the blood vessels and affect cancer cells, angiogenesis inhibitors have the advantage of facilitating drug delivery by directly contacting vascular endothelial cells.

[0005] Inhibiting angiogenesis is a fundamental treatment option for diseases associated with excessive angiogenesis, such as cancer, ophthalmology, arthritis, and psoriasis. However, currently available angiogenesis inhibitors are generally manufactured through organic synthesis, resulting in unsatisfactory efficacy and serious side effects. Consequently, research is urgently needed to develop angiogenesis inhibitors with high pharmacological properties and minimal side effects.

[0006] Meanwhile, there are two strategies for inhibiting angiogenesis: one that interferes with signals that induce angiogenesis, and the other that activates anti-angiogenic signals using angiogenesis inhibitors such as pigment epithelial-derived factor (PEDF).

[0007] Among them, PEDF 34-mer has the limitation of having a short half-life as a peptide drug, and thus, no research has been reported to date on using PEDF 34-mer or short peptide-based nanostructures derived from PEDF 34-mer as drug delivery vehicles.

[0008] Accordingly, the present inventors have completed the present invention to provide a fusion protein as a drug delivery vehicle for inhibiting angiogenesis using the self-assembly function of EBP and a complex action including the activation function of the anti-angiogenic mechanism of PEDF 34-mer, and a nanostructure comprising the fusion protein.

[0009]

[0010] The technical problem to be achieved by the present invention is to provide a multi-stimulus responsive block copolypeptide that sensitively responds to temperature and metal ion stimulation.

[0011] In addition, the present invention aims to provide a nanostructure formed by self-assembly of the above multi-stimulus responsive block copolypeptide as a drug delivery vehicle.

[0012] In addition, the present invention aims to provide a pharmaceutical composition for treating diseases caused by angiogenesis by fusing a peptide having an angiogenesis inhibitory function to the above fusion polypeptide.

[0013] However, the technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0014] The present invention provides a multi-block copolypeptide comprising a peptide derived from pigment epithelial-derived factor (PEDF) and an elastin-based polypeptide (EBP), and is represented by the following formula 1.

[0015] [Formula 1]

[0016] [A]-[B]n-[C]m-[B]n-[A]

[0017] In the above equation 1,

[0018] The above A block is a PDEF-derived peptide,

[0019] The above B block or C block are different EBPs,

[0020] n and m are integers that are equal or different and represent the number of times each block is repeated.

[0021] In addition, the present invention provides a multi-stimulus responsive drug delivery system comprising a multi-block copolypeptide of any one of the above-mentioned items and exhibiting stimuli responsiveness.

[0022] In addition, the present invention provides a pharmaceutical composition comprising a multi-block copolypeptide comprising a peptide derived from an epithelial-derived factor (PEDF); and an elastin-based polypeptide (EBP).

[0023] A pharmaceutical composition represented by the following formula 1 is provided.

[0024] [Formula 1]

[0025] [A]-[B]n-[C]m-[B]n-[A]

[0026] In the above equation 1,

[0027] The above A block is a PDEF-derived peptide,

[0028] The above B block is a hydrophobic EBP,

[0029] The above C block is a hydrophilic EBP,

[0030] n and m are integers that are equal or different and represent the number of times each block is repeated.

[0031] In addition, a pharmaceutical composition for inhibiting angiogenesis is provided, characterized in that it prevents or treats a disease caused by angiogenesis, including the pharmaceutical composition.

[0032] The present invention comprises a multi-stimulus responsive block copolypeptide comprising a histidine-containing EBP triblock copolypeptide comprising an antiangiogenic peptide at both ends, which is His-Zn 2+ Self-assembly into multivalent nanostructures with controlled size through ignition can exhibit enhanced antiangiogenic activity.

[0033] The above nanostructure can suppress the migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner under angiogenic stimulation induced by vascular endothelial growth factor (VEGF), and since the nanostructure containing ZnCl₂ exhibits a higher anti-angiogenic effect, it can be utilized as an anti-angiogenic therapeutic agent.

[0034] To facilitate a more thorough understanding of the drawings cited in the detailed description of this application, a brief description of each drawing is provided.

[0035] Figure 1 (A) shows the gene and amino acid sequences of PEDF 34-mer peptides, EBP[A₁₄I₁]₁, and EBP[H₃A₂G₁]₁. (B) shows the PEDF 34-mer-EBP[H₃A₂G₁]-EBP[A₁G₄I₁]-EBP[H₃A₂G₁]-PEDF 34-mer (PEDF-E n-6-n -PEDF) block design schematic. (C) PEDF-E in the presence and absence of ZnCl₂ n-6-n -PEDF is a self-assembled nanostructure. Above the transition temperature (Tt), the amphoteric PEDF-E n-6-n-PEDF self-assembles to form vesicular structures, which decrease in size when ZnCl₂ is added. (D) Fluorescence microscopy images of HUVECs: (a) without VEGF, (b) with 50 ng / ml VEGF, and (c) with 50 ng / ml VEGF and 1000 nM PEDF-E. 12-6-12 -PEDF treatment, (d) 50 ng / ml VEGF, 1000 nM PEDF-E 12-6-12 -Evaluation of antiangiogenic effect by treatment with PEDF and 9000 nM ZnCl₂. (Scale bar: 500 μm).

[0036] Figure 2 (A) is an agarose gel electrophoresis image of a PEDF-EBP triblock copolypeptide. (B) is an SDS-PAGE image of a PEDF-EBP triblock copolypeptide. The lengths of the gene fragments are indicated below the corresponding fragments, and the expected molecular weights are shown below the bands. The SDS-PAGE gel was visualized by copper staining. (C-F) is a PEDF-E 6-6-6 -PEDF and PEDF-E 12-6-12 -Turbidity profiles of PEDF at concentrations of 10–100 μM at pH 6.4–8.0. (C) PEDF-E at concentrations of 10–100 μM in 10 mM PBS. 6-6-6 -PEDF, (E) PEDF-E 12-6-12 -Turbidity profile of PEDF. (D) PEDF-E at pH 6.4 - 8.0 6-6-6 -PEDF, (F) PEDF-E 12-6-12 -Turbidity profile of PEDF.

[0037] Figure 3 (A) shows 10 μM E in 10 mM HEPES 6-6-6 , E 12-6-12 , PEDF-E 6-6-6 -PEDF and PEDF-E 12-6-12 - Turbidity profile of PEDF. (B) PEDF-E according to ZnCl₂ concentration (0, 45, 90, 180 μM) in 10 mM HEPES 12-6-12-Turbidity profiles of PEDF (measured at 350 nm with a heating rate of 1°C / min). (C, D) 10 μM PEDF-E in the presence of 10 mM HEPES and ZnCl₂ 12-6-12 -The hydration radius of PEDF (R H , Hydrodynamic radius) measured at: (C) 20°C, (D) 37°C. H .

[0038] Figure 4 (A, B) is PEDF-E 12-6-12 -R of PEDF nanostructures H and FITC-fused PEDF-E 12-6-12 -CLSM images of PEDF nanostructures. (A-D) CLSM images taken after dialysis in deionized water (DW). Scale bar: 8 μm. (E-H) CLSM images taken after dialysis in 10 mM PBS. Scale bar: 4 μm. (I and J) 10 μM PEDF-E with or without ZnCl₂. 12-6-12 -CLSM images of PEDF: (I) without ZnCl₂. Scale bar: 5 μm and scale bar of enlarged image: 2 μm. (J) in the presence of 90 μM ZnCl₂. (K and L) with 10 μM PEDF-E in the absence of ZnCl₂. 12-6-12 -TEM image of PEDF: TEM sample dried at 37°C. (Scale bars: 1 μm and 200 nm).

[0039] Figure 5 shows PEDF-E with or without ZnCl₂. 12-6-12 -The in vitro antiangiogenic effect of PEDF is shown. (A) is a fluorescence microscope image. (B) shows the degree of tube formation in HUVECs quantified based on the image in (A). HUVECs stained with Calcein-AM were cultured on Matrigel for 4 hours, and cell migration was induced with 50 ng / ml VEGF, and 0.01–1000 nM PEDF-E was added. 12-6-12- Treated with PEDF (with or without ZnCl₂). The ZnCl₂ concentration was adjusted to 9 times the polypeptide concentration. (Scale bar: 500 μm).

[0040] Figure 6 is PEDF-E 12-6-12 -The in vitro antiangiogenic effect of PEDF according to its structure is shown. (A) is a fluorescence microscopy image. (B) is the normalized tube length of HUVECs quantified based on the image in (A). HUVECs stained with Calcein-AM were cultured on Matrigel for 4 hours, and cell migration was induced with 50 ng / ml VEGF. 0.001, 1, and 1000 nM PEDF-E 12-6-12 -PEDF and ZnCl₂ containing group were prepared in HEPES to induce the formation of aggregates (coacervate), and 0.001 - 1000 nM PEDF-E 12-6-12 -PEDF was prepared in PBS to induce the formation of toroidal structures. (Scale bar: 500 μm).

[0041] Figure 7 is PEDF-E 12-6-12 -The in vitro antiangiogenic effect of PEDF according to its structure (same experiment as in Figure 6) is shown. (A) is a fluorescence microscope image. (B) is the normalized number of junctions of HUVECs quantified based on the image in (A). HUVECs stained with Calcein-AM were cultured on Matrigel for 4 hours, and cell migration was induced with 50 ng / ml VEGF. 0.001, 1, and 1000 nM PEDF-E 12-6-12 -PEDF and ZnCl₂ containing groups were prepared in HEPES to induce aggregate formation, and 0.001 - 1000 nM PEDF-E 12-6-12 -PEDF was prepared in PBS to induce the formation of toroidal structures. (Scale bar: 500 μm) .

[0042] Figure 8 is E 12-6-12 and PEDF-E 12-6-12-The cytotoxicity evaluation of PEDF is shown. 0.01 - 1000 nM E according to the presence or absence of ZnCl₂ 12-6-12 and PEDF-E 12-6-12 -After PEDF treatment for 4 and 12 hours, cell viability was quantified using the WST-1 assay. The ZnCl₂ concentration was adjusted to 9 times the polypeptide concentration.

[0043]

[0044] The terminology used in this specification is for the purpose of describing embodiments and is not intended to limit and / or restrict the present application. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, it should be understood that the terms "comprises" or "has" are intended to indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0045]

[0046] The term "amino acid" in the present invention refers to a natural amino acid or an artificial amino acid, and preferably refers to a natural amino acid. For example, the amino acid refers to glycine, alanine, serine, valine, leucine, isoleucine, methionine, glutamine, asparagine, cysteine, histidine, phenylalanine, arginine, tyrosine, or tryptophan. The properties of the amino acids are widely known in the art. Specifically, they exhibit hydrophilicity (negative or positive charge) or hydrophobicity, and also exhibit aliphatic or aromatic properties.

[0047] Abbreviations used in this specification, such as Gly (G) and His (H), are amino acid abbreviations. Gly is an abbreviation for glycine, and His is an abbreviation for histidine. Glycine is also expressed as G, and histidine is also expressed as H. The above abbreviations are widely used in this technical field.

[0048] As used herein, the term "polypeptide" refers to any polymer chain of amino acids. The terms "peptide" and "protein" may be used interchangeably with the term "polypeptide," which also refers to a polymer chain of amino acids. The term "polypeptide" encompasses natural or synthetic proteins, protein fragments, and polypeptide analogs of protein sequences. Polypeptides may be monomers or polymers.

[0049] In the present invention, the term 'copolypeptide' means a polypeptide that is a copolymer.

[0050] The copolypeptide of the present invention basically comprises elastin-based polypeptides (EBPs), and the 'elastin-based polypeptides' are also called 'elastin-like polypeptides (ELPs).' This is a term widely used in the technical field of the present invention.

[0051] The term "pharmaceutical composition" in the present invention refers to a composition manufactured for the purpose of preventing or treating a disease, and may be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0052]

[0053] Multi-stimulus responsive block copolypeptide

[0054] One aspect of the present invention provides a multi-block copolypeptide comprising a peptide derived from epithelial-derived factor (PEDF); and an elastin-based polypeptide (EBP).

[0055] A multi-block copolypeptide represented by the following formula 1.

[0056] [Formula 1]

[0057] [A]-[B]n-[C]m-[B]n-[A]

[0058] In the above equation 1,

[0059] The above A block is a PDEF-derived peptide,

[0060] The above B block or C block are different EBPs,

[0061] n and m are integers that are equal or different and represent the number of times each block is repeated.

[0062] The above multiblock copolypeptide comprises a histidine-containing EBP triblock copolypeptide containing an antiangiogenic peptide at both ends, which is His-Zn 2+ Self-assembly into multivalent nanostructures with controlled size through ignition can exhibit enhanced antiangiogenic activity.

[0063] The above EBP triple block is composed of a hydrophilic EBP having a transition temperature (Tt) higher than body temperature and a hydrophobic EBP containing histidine, and His-Zn 2+ Self-assembles into size-controlled ABA-type nanostructures through ignition. The PEDF 34-mer, an antiangiogenic 34-mer domain of pigment epithelium-derived factor (PEDF), can be introduced at both ends of the EBP triblock to impart multivalent properties.

[0064] The above PEDF-derived peptide may comprise the amino acid sequence of SEQ ID NO: 3.

[0065] Sequence number 3: DPFFK VPVNK LAAAV SNFGYDLYR VRSST SPTTN

[0066] In one embodiment of the present invention, an EBP triblock copolypeptide comprising an antiangiogenic peptide at both ends thereof is provided, wherein the peptide can form a nanostructure of controlled size through self-assembly to enhance antiangiogenic activity. The block copolypeptide is composed of an antiangiogenic 34-mer domain of pigment epithelium-derived factor (PEDF) and a thermosensitive elastin-based polypeptide (EBP) containing histidine, which comprises a histidine-Zn 2+ It can self-assemble into size-controllable nanostructures through ignition.

[0067] The above nanostructures can inhibit the migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner under angiogenic stimulation induced by vascular endothelial growth factor (VEGF), and a higher anti-angiogenic effect can be exhibited in small nanostructures containing ZnCl₂ than in large nanostructures not containing ZnCl₂.

[0068]

[0069] elastin-based polypeptide (EBP)

[0070] One aspect of the present invention may be a method in which the PEDF-derived peptide is linked to both ends of a hydrophobic elastin-based polypeptide. The hydrophobic EBP may continuously and repeatedly comprise a block comprising the amino acid sequence of SEQ ID NO: 1. Specifically, the number of repetitions of the hydrophobic EBP block may be 1 to 36.

[0071] Sequence number 1: IPAHGIPAAGIPAHGIPAIGIPAHGIPAAG

[0072] The hydrophilic EBP may continuously and repeatedly include a block consisting of an amino acid sequence of SEQ ID NO: 2. The number of repetitions of the hydrophilic EBP block may be 1 to 36.

[0073] Sequence number 2: VPAGG VPAAG VPAGG VPAGG VPAI

[0074] EBP block copolypeptides have different transition temperatures (Tt) for each EBP block, and exhibit amphiphilic properties in the temperature range between the Tts of each block and self-assemble into nanostructures such as micelles and vesicles. Self-assembled nanostructures of EBP can have additional functions or sensitivity by utilizing amino acids at the Xaa position. For advanced drug delivery, histidine was introduced as a guest moiety to provide additional functionality, and the imidazole side chain of histidine has a pKa of 6, Cu 2+ , Zn 2+ , Mg 2+ ², Ni 2+ , Co 2+ , Cd 2+ It can complex with metal ions such as:

[0075] Histidine-rich EBPs are pH-sensitive due to the reversible charge change of their imidazole side chains, which induces a change in Tt as they change from positive at low pH to neutral at high pH. Self-assembled EBP nanostructures containing histidine in the hydrophobic core have been applied for tumor-targeted drug delivery through pH-sensitive disassembly. Furthermore, self-assembly of EBPs containing histidine in the hydrophobic core is affected not only by pH but also by metal ions. Introducing metal ions to the histidine in EBPs decreases the critical micelle concentration (CMC) of the self-assembled structures, makes the EBP core more compact, and consequently, indicates that the stability of EBP nanostructures is enhanced by the metal ion-containing histidine.

[0076] Various EBP triblock copolypeptides in the ABA or BAB form have been designed and studied for the development of hollow spheres, vesicles, or hydrogels. Furthermore, EBP triblock copolypeptides in the ABC form, composed of EBP diblock copolypeptides and other polypeptides such as coiled-coil proteins or cross-linking domains, have been utilized in the study of advanced nanostructures.

[0077] Furthermore, therapeutic protein C was introduced at the hydrophilic end and expressed in a multivalent shell form. Previous studies have shown that nanostructures containing multivalent C, utilizing ligand-receptor or antigen-antibody binding affinity, exhibited enhanced efficacy compared to single therapeutic proteins. Therefore, EBP block copolypeptides in the form of ABA, BAB, and ABC can be utilized as promising biomaterials for advanced drug delivery systems in combination with other functional proteins or amino acids.

[0078] The EBP according to the present invention may be a polypeptide in which a pentapeptide is repeated, and the repeated polypeptide may form a polypeptide block (EBP block).

[0079] EBPs undergo a reversible phase transition at their lower critical solution temperature (LCST), also called the transition temperature (Tt). They are highly soluble below Tt, but become insoluble above Tt.

[0080] In the present invention, the X is referred to as a 'guest moiety'. Various types of EBPs according to the present invention can be manufactured by introducing the X in various ways. In particular, the physicochemical properties of the EBP can be controlled according to the critical solution temperature (LCST) of the X.

[0081]

[0082] Peptide derived from epithelial-derived factor (PEDF)

[0083] In one aspect of the present invention, the multi-stimulus responsive block copolypeptide may comprise a PEDF-derived peptide, and the PEDF-derived peptide may comprise the amino acid sequence of SEQ ID NO: 3.

[0084] Pigment epithelium-derived factor (PEDF) structurally belongs to the serpin superfamily, but it does not exhibit the structural changes seen in serpins while inhibiting serine proteases involved in various biological processes. PEDF activates antiangiogenic signaling and acts as an angiogenesis inhibitor, and has additional properties such as neurotrophic and antitumor activity, but does not exhibit serine protease inhibitory activity.

[0085] A 34-mer peptide (residues 44-77) with antiangiogenic properties has been identified from PEDF, and its associated mechanisms have been elucidated. While associations between PEDF 34-mer and intracellular signaling pathways related to apoptosis and antiangiogenesis have been established, some of the mechanisms remain to be fully elucidated. PEDF 34-mer can induce apoptosis by activating c-Jun-NH2 kinase (JNK) and exert antiangiogenic effects by inactivating nuclear factor of activated T cells (NFAT).

[0086] PEDF 34-mer binds with affinity to the catalytic beta subunit of F1-ATP synthase on the surface of endothelial and tumor cells, inhibiting ATP synthase activity and inhibiting tumor growth and invasion. Furthermore, PEDF 34-mer binds to laminin receptors on endothelial cell membranes, inducing endothelial cell death and inhibition of migration.

[0087] In addition to these mechanistic studies, narrower antiangiogenic peptide regions were identified within the PEDF 34-mer, and their antiangiogenic activities and mechanisms were analyzed in comparison with the PEDF 34-mer.

[0088]

[0089] Self-assembled nanostructures and drug delivery systems

[0090] Triblock copolymers and their self-assembled nanostructures have been extensively studied for biomedical applications due to their tunable properties and diverse structures. Triblock copolymers consist of hydrophobic and hydrophilic polymers (represented by A and B), which impart amphiphilic properties. Additionally, a variable polymer (represented by C) is included to introduce additional functionality. Each polymer can be systematically combined and arranged to design various triblock copolymers, such as ABA, BAB, and ABC, which can be used to fabricate diverse nanostructures, such as micelles and vesicles. The properties and functions of the A and B polymers are tailored for self-assembly of nanostructures and biomedical applications.

[0091] The properties and functions of polymers A and B can be tailored to self-assemble into nanostructures and be applied as biomedical materials. For example, stimuli-responsive polymers are used as advanced drug delivery carriers, controlling drug loading and release by altering their structure in low-pH environments, such as tumors. Furthermore, polymers containing metal or drug-binding ligands are incorporated into the hydrophobic portion of self-assembled nanostructures through binding or coordination with metals and drugs.

[0092] For applications as therapeutics as well as drug delivery carriers, therapeutic domains or receptor-targeting ligands are incorporated into polymers A and B. The composition and length of biopolymer polypeptides can be more precisely and precisely controlled through genetic synthesis than with synthetic polymers, enabling the creation of monodisperse nanostructures.

[0093] As drug delivery vehicles, stimuli-responsive polymers are used to control drug loading and release by changing their structure in target environments, such as the low pH of tumors.

[0094] In one embodiment of the present invention, the stimulus is temperature, protein concentration, pH or Zn 2+ The ions may be one or more. The copolypeptide may form a nanostructure whose size is controlled by the stimulus. Specifically, the nanostructure may be a coacervate or toroidal structure.

[0095]

[0096] Additionally, polymers containing metal- or drug-binding ligands are incorporated into the hydrophobic portion of self-assembled nanostructures, where they are assembled through binding to metals or drugs. For use not only as therapeutics but also as drug delivery vehicles, therapeutic domains or receptor-targeting ligands have been incorporated into polymers A and B. Polypeptides, as biopolymers, allow for more precise control of their composition and length through genetic synthesis than synthetic polymers, enabling the creation of monodisperse nanostructures.

[0097]

[0098] In another aspect of the present invention, a pharmaceutical composition is provided comprising a multi-block copolypeptide comprising a peptide derived from epithelial-derived factor (PEDF); and an elastin-based polypeptide (EBP).

[0099] A pharmaceutical composition represented by the following formula 1.

[0100] [Formula 1]

[0101] [A]-[B]n-[C]m-[B]n-[A]

[0102] In the above equation 1,

[0103] The above A block is a PDEF-derived peptide,

[0104] The above B block is a hydrophobic EBP,

[0105] The above C block is a hydrophilic EBP,

[0106] n and m are integers that are equal or different and represent the number of times each block is repeated.

[0107] The pharmaceutical composition of the present invention may additionally contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, salts for osmotic pressure control or buffers, and other therapeutically useful substances, and may be formulated according to conventional methods.

[0108] The pharmaceutical composition according to the present invention can be administered via various routes, including oral, transdermal, subcutaneous, intravenous, or intramuscular. The dosage of the active ingredient can be appropriately selected based on various factors, such as the route of administration, the patient's age, sex, weight, and severity of the condition. Furthermore, the composition of the present invention can be administered in combination with known compounds capable of enhancing the desired effect.

[0109] In addition, in another aspect of the present invention, a pharmaceutical composition for inhibiting angiogenesis is provided, characterized in that it prevents or treats a disease caused by angiogenesis, including the pharmaceutical composition.

[0110] In the present invention, "angiogenesis-related disease" or "disease caused by angiogenesis" refers to a disease caused by abnormal progression of angiogenesis. Throughout this specification, the terms "angiogenesis-related disease" and "disease caused by angiogenesis" may be used interchangeably.

[0111] Angiogenesis-related diseases that can be prevented or treated by the composition of the present invention may include angiogenesis-dependent cancer, benign tumors, rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, proliferative retinopathy, erythroderma, psoriasis, Osler-Weber syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasias, hemophilic arthropathy, angiofibromas, traumatic granulation, intestinal adhesions, dialysis graft vascular access stenosis, arteriosclerosis, scleroderma, hypertrophic scars, cat scratch disease, Helicobacter pylori ulcers, obesity, or inflammation. The composition of the present invention can effectively prevent or treat the above angiogenesis-related diseases by inhibiting angiogenesis or vasculogenesis.

[0112]

[0113] Hereinafter, exemplary embodiments according to the present application will be described in detail with reference to the contents described in the attached drawings, but the scope of the present invention is not limited by the contents presented below.

[0114] ingredient

[0115] BL21 (DE3) Escherichia coli (E. coli) cells and pET-21a (+) vector were provided by Novagen Inc., Madison, WI, USA. Top10 competent cells were purchased from Invitrogen, Carlsbad, CA, USA.

[0116] All custom oligonucleotides were synthesized by Cosmo Gene Tech, Seoul, South Korea. Calf intestinal alkaline phosphatase (CIP), BamHI, and XbaI were provided by Thermo Fisher Scientific, Waltham, MA, USA, and AcuI and BseRI were purchased from New England Biolabs, Ipswich, MA, USA. T4 DNA ligase was provided by Elpis Biotech, Daejeon, South Korea.

[0117] DNA miniprep, gel extraction, and PCR purification kits were provided by Geneall Biotechnology, Seoul, Korea, and Dyne Agarose High was purchased from DYNE BIO, Inc., Seongnam, Korea.

[0118] Top10 cells and BL21(DE3) cells were cultured in Circle Grow medium provided by MP Biomedicals, Solon, OH, USA. Isopropyl-D-thiogalactoside (IPTG) was supplied by Goldbio, St. Louis, MO, USA.

[0119] Ampicillin, polyethyleneimine (PEI), phosphate-buffered saline (PBS, pH 7.4), phosphotungstic acid (PTA), and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich, St. Louis, MO, USA.

[0120] Matrigel was purchased from BD Biosciences, San Diego, CA. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza, Basel, Switzerland, and cultured using the Endothelial growth medium-2 (EGM-2) Bullet Kit containing Endothelial basal medium-2 (EBM-2).

[0121] Calcein-AM was purchased from Invitrogen, Carlsbad, CA, USA, and recombinant human vascular endothelial growth factor-165 (rhVEGF165) was provided by R&D SYSTEMS, Minneapolis, MN, USA.

[0122] Cell Counting Kit-8 (CCK-8) was supplied by Dojindo Laboratories, Kumamoto, Japan.

[0123]

[0124] Manufacturing examples: Gene construction and protein expression and purification

[0125] Plasmids containing PEDF-EBP triblock copolypeptides were constructed by inserting the PEDF 34-mer gene into the EBP triblock copolypeptide gene. Briefly, oligonucleotide pairs encoding each EBP[A₁G₄I₁]₁ and EBP[H₃A₂G₁]₁ were annealed, and the annealed genes were iteratively inserted into modified pET21a (mpET21a), which was constructed according to a previously reported method. The EBP[H₃A₂G₁] genes in the plasmid were constructed using a modified repetitive directed ligation (RDL) method.

[0126] The PEDF 34-mer gene was constructed by PCR amplifying the region 130-231 of the human PEDF cDNA sequence, digesting the region with XbaI and NheI, and inserting the resulting region into pET21a. The PEDF-EBP triblock copolypeptide gene was constructed by sequentially inserting the EBP gene fragment digested with XbaI and AcuI and the PEDF 34-mer gene fragment digested with AcuI into mpET21a, which had been digested with XbaI, BseRI, and CIP and dephosphorylated. All cloning processes were confirmed by agarose gel electrophoresis and DNA sequencing after digestion with XbaI and BamHI.

[0127] The PEDF-EBP triblock copolypeptide was expressed in Escherichia coli (E. coli) and purified using reverse phase transfer (ITC). The plasmid containing the protein gene was transformed into BL21(DE3) strain E. coli, and the transformed E. coli was cultured in CircleGrow medium containing 50 μg / mL ampicillin. When the 600 nm absorbance of the culture medium reached 1.0, protein expression was induced by the addition of IPTG (final concentration of 1 mM). The expressed protein was overexpressed overnight at 37°C and 200 rpm and harvested by centrifugation (4500 rpm, 10 min, 4°C).

[0128] Harvested cells were resuspended in 0.01 M PBS and lysed by sonication (5 min total, 20 s pauses with 10 s intervals). 0.5% w / v PEI was then added and centrifuged (13,000 rpm, 15 min, 4°C) to remove nucleic acids.

[0129] Phase transition of the protein solution was induced by adding 3 M sodium chloride (NaCl) and heating to 40°C, and the aggregated EBP was pelleted by centrifugation (13,000 rpm, 15 min, 40°C). The separated aggregates were resuspended in 0.01 M PBS at 4°C, and insoluble protein contaminants were removed by centrifugation (13,000 rpm, 15 min, 4°C). This aggregation and resuspension process was repeated until the protein was purified.

[0130]

[0131] Experimental Example 1: Analysis of thermal sensitivity and self-assembly characteristics

[0132] To analyze the thermal sensitivity, the absorbance at 350 nm of PEDF-EBP triblock copolypeptide in solutions containing 0.01 M HEPES and ZnCl₂ was measured using a Cary 100 Bio UV / Vis spectrophotometer (Varian Instruments, Walnut Creek, CA) over the range of 20–60°C at a heating rate of 1°C / min. The transition temperature (Tt) was defined as the inflection point of the thermal profile.

[0133] The thermally induced self-assembly properties were determined by measuring the hydration radius (R) of the protein at a concentration of 10 μM in a solution containing 0.01 M HEPES and ZnCl₂ using a Nano ZS90 (ZEN3690) dynamic light scattering (DLS) instrument (Malvern Instruments, Worcestershire, UK). H ) was analyzed by measuring at 20°C and 37°C.

[0134]

[0135] Experimental Example 2: Imaging in Swollen and Dry States

[0136] 100 μM protein was incubated with 100 μM FITC in deionized water at 4°C for 4 h to induce binding, and then unbound FITC was removed by dialysis. Self-assembled protein structures in the swollen state were imaged using confocal laser scanning microscopy (CLSM). Proteins were prepared in deionized water, 10 mM PBS, or 10 mM HEPES, and images were taken at 37°C using a Zeiss LSM800 (Leica, Wetzlar, Germany) and a CU302 temperature controller (LCI, Namyangju, South Korea) in the absence and presence of ZnCl₂, respectively.

[0137] Self-assembled protein structures in the dry state were analyzed using transmission electron microscopy (TEM). Proteins were prepared in 0.01 M solutions in the absence and presence of ZnCl₂, dried on carbon-coated grids at 37°C, and then TEM samples were prepared. Images of the dried structures were acquired using a JEM-2100F FE-STEM (JEOL, Freising, Germany) operating at 200 kV.

[0138]

[0139] Experimental Example 3: PEDF-E in a test tube 12-6-12 -Analysis of the inhibitory effect of HUVEC tube formation according to the type of PEDF self-assembly structure.

[0140] PEDF-E according to the type of self-assembled structure 12-6-12 -The inhibitory effect of PEDF on HUVEC tube formation was evaluated in vitro.

[0141] PEDF-E in the absence of ZnCl₂ 12-6-12 -PEDF forms a vesicle structure, and in the presence of ZnCl₂, PEDF-E 12-6-12-PEDF forms coacervates. Calcein-stained HUVECs (2 Х 10⁴ cells / well) were cultured on Matrigel with 50 ng / mL rhVEGF165 and PEDF-EBP triblock copolypeptides at concentrations ranging from 0.01 to 1,000 nM in the absence and presence of ZnCl₂ (9-fold protein concentration) for 4 h at 37°C.

[0142] The tube structures of HUVECs on Matrigel were imaged using an Axio Observer A1 microscope (ZEISS, Oberkochen, Germany), and the tube length was measured using the angiogenesis analyzer of Image J Lab software.

[0143] Fluorescence microscopy images of HUVECs stained with Calcein-AM for 4 hours are shown in Fig. 5(A), and the extent of tube formation is graphically represented in Fig. 5(B) based on the normalized tube length of HUVECs.

[0144] PEDF-E at 0.001, 1, and 1000 nM 12-6-12 -PEDF (with or without ZnCl₂) was prepared in HEPES to form vesicles or aggregates (coacervate), and PEDF-E in the range of 0.001 - 1000 nM 12-6-12 -PEDF was prepared in PBS to form a toroidal structure.

[0145] These constructs were treated with 50 ng / mL rhVEGF165 to induce migration and tube formation in HUVECs.

[0146] Fluorescence microscopy images of HUVECs stained with Calcein-AM for 4 hours are shown in Figures 6(A) and 7(A), and the extent of tube formation was graphically represented in Figures 6(B) and 7(B) based on the normalized tube length or number of junctions of HUVECs.

[0147] In Figures 6 (A) and 7 (A), HUVECs treated with rhVEGF165 migrated more and formed networks than those treated without rhVEGF165.

[0148] After setting the tube length or number of junctions to 0% and 100% in the absence and presence of VEGF, respectively, PEDF-E 12-6-12 -PEDF treatment groups were normalized and compared.

[0149]

[0150] Experimental Example 4: PEDF-E 12-6-12 -Cytotoxicity evaluation of PEDF structures

[0151] Previous studies have reported that PEDF 34-mer reduces cell viability through ATP synthase inhibition and is involved in various apoptotic processes.

[0152] Accordingly, PEDF-E 12-6-12 -To evaluate the cytotoxicity of each structure according to the presence or absence of ZnCl₂ in PEDF, the HUVEC viability was quantified using the WST-8 reagent, and the results are shown in Fig. 8.

[0153] 3 Х10³ cells / well of HUVECs were seeded in a 96-well plate and cultured overnight at 37°C to induce attachment. Afterwards, 100 μL of PEDF-E at concentrations ranging from 0.01 to 1000 nM was added. 12-6-12 -After PEDF (with or without ZnCl₂, ZnCl₂ concentration: 9 times the protein concentration) was treated on HUVEC, the cells were cultured for 4 and 12 hours to check the change in cell viability.

[0154] ZnCl₂ alone as a control, E 12-6-12 (Including the presence or absence of ZnCl₂, ZnCl₂ concentration: 9 times the protein concentration) was used.

[0155] After incubation, the medium containing the protein was removed, 100 μL of 10% water-soluble tetrazolium salt (WST-8) was added to EBM-2 medium, and the cells were further incubated at 37°C for 1 to 4 hours.

[0156] The color change of the medium according to cell viability was quantified by measuring the absorbance at 450 nm using a Synergy MX Microplate Reader (BioTek, Winooski, USA).

[0157] When HUVECs were maintained in culture medium, the survival rate was set to 100%, and the survival rates of other experimental groups were normalized and graphed in Figure 8.

[0158]

[0159]

[0160] Results and Discussion

[0161] Zn 2+ We aimed to enhance antiangiogenic function by designing self-assembled nanostructures containing multivalent antiangiogenic peptides whose size can be controlled through histidine interactions.

[0162] The designed antiangiogenic nanostructure consists of an antiangiogenic PEDF 34-mer and a thermoresponsive EBP triblock copolypeptide consisting of a hydrophilic EBP block and a hydrophobic histidine-rich EBP block, which is designed to enable stimuli-responsive self-assembly and structure size control through His-Zn² complexation at body temperature. The EBP triblock copolypeptide containing PEDF 34-mer (PEDF-EBP triblock copolypeptide) was constructed by fusing the designed EBP gene with a PEDF 34-mer gene fragment PCR-amplified from PEDF cDNA.

[0163] Figure 1(A) shows the genetic and amino acid sequences of the PEDF 34-mer, the hydrophilic EBP block (EBP[A₁G₄I₁]₁), and the hydrophobic EBP block (EBP[H₃A₂G₁]₁) in the EBP triblock copolypeptide. The transition temperature (Tt) of the EBP was adjusted according to the repeating pentapeptide sequence, guest residues, their ratio, and block length. The hydrophilic EBP block was designed to have a Tt higher than 37°C and did not contain charged amino acids to minimize the influence on the PEDF 34-mer. The hydrophilic EBP block EBP[A₁G₄I₁]n was composed of a structure of six repeating pentapeptides (VPAXG) with guest residues and their ratio (A:G:I = 1:4:1).

[0164] The hydrophobic EBP block is designed to have a Tt below 37°C, and Zn 2+ Metal ion reactivity was made to control the size of self-assembled nanostructures through interaction with hydrophobic EBP block EBP[H₃A₂G₁]n, which is composed of six pentapeptides (IPAXG), and the ratio of guest residues was adjusted to H:A:G = 3:2:1.

[0165] Figure 1(B) shows a PEDF-EBP triblock copolypeptide, i.e., PEDF 34-mer-EBP[H₃A₂G₁]n-EBP[A₁G₄I₁]6-EBP[H₃A₂G₁]n-PEDF 34-mer (PEDF-E n-6-n -PEDF) schematic diagram. PEDF 34-mer was introduced at both ends of EBP triblock copolypeptide for multivalent exposure on the surface of self-assembled nanostructures.

[0166] PEDF-E without and with ZnCl₂ respectively n-6-n-PEDF self-assembly process and formed structures are presented with images in Fig. 1(C). Above Tt, hydrophobic EBP, EBP[H₃A₂G₁]n, was hydrophobic, whereas EBP[A₁G₄I₁]n still maintained hydrophilicity. Accordingly, PEDF-E with amphiphilic properties n-6-n -PEDF self-assembled into vesicular structures containing multivalent PEDF 34-mers of 2 μm or more.

[0167] Zn 2+ PEDF-E included n-6-n -PEDF changed from a soluble monomer to a multimer below Tt, and above Tt, an unstable amphiphilic structure self-assembled into an aggregate (coacervate) of less than 1 μm.

[0168] Zn 2+ PEDF-E according to ignition n-6-n -The antiangiogenic activity of the PEDF construct was evaluated through in vitro HUVEC tube formation experiments. Figure 1(D) shows a fluorescence microscopy image of HUVEC on Matrigel.

[0169] (a) without VEGF, (b) with 50 ng / mL VEGF, (c) with 50 ng / mL VEGF and 1000 nM PEDF-E 12-6-12 -If PEDF is included, (d) 50 ng / mL VEGF, 1000 nM PEDF-E 12-6-12 -Images are shown when PEDF and 9000 nM ZnCl₂ are included.

[0170] VEGF-stimulated HUVECs migrated and formed tube-like structures. VEGF-induced HUVEC migration and tube formation were significantly enhanced in the absence and presence of ZnCl₂, respectively, by PEDF-E. 12-6-12 -Evaluated by PEDF nanostructures.

[0171]

[0172] E n-6-n and PEDF-E n-6-n -PEDF (n = 6 and 12) gene composition and expression

[0173] E for expression in E. coli n-6-n and PEDF-E n-6-n -The PEDF gene was constructed by sequentially inserting the EBP and PEDF 34-mer genes into the mpET21 vector. Gene cloning through this sequential insertion was confirmed by treatment with XbaI and HindIII restriction enzymes, agarose gel electrophoresis, and DNA sequencing.

[0174] E treated with restriction enzymes XbaI and HindIII n-6-n and PEDF-E n-6-n -The agarose gel electrophoresis results of the PEDF gene are shown in Figure 2(A). The sizes of the restricted DNA fragments are indicated at the bottom of the gel, and the DNA standard markers are shown on the left.

[0175] The size of the DNA limiting in the agarose gel is E n-6-n and PEDF-E n-6-n -It was 82 base pairs (bp) longer than the sequence encoding the PEDF gene, because the XbaI and HindIII restriction enzyme cleavage sites were located outside the gene region. In addition, PEDF-E n-6-n -The length of the PEDF gene is E n-6-n It was 105 bp longer than the 102 bp long PEDF 34-mer gene and the 3 bp sticky end added at the restriction enzyme cleavage and ligation sites.

[0176] E n-6-n and PEDF-E n-6-n-PEDF protein was expressed in E. coli and purified using the same reverse phase transfer (ITC) method as the temperature-sensitive EBP purification method reported in a previous study. The purity and molecular weight of the purified protein were confirmed by SDS-PAGE using copper staining, and the results are shown in Figure 2(B). The expected E n-6-n and PEDF-E n-6-n -The molecular weight of PEDF is indicated at the bottom of each protein band.

[0177] PEDF-E n-6-n -The molecular weight of PEDF is E n-6-n The size of PEDF 34-mer increased by 3763.21 Da, as confirmed in the SDS-PAGE image.

[0178]

[0179] PEDF-E n-6-n -Thermosensitivity of PEDF and changes according to concentration and pH

[0180] PEDF-E n-6-n The results of analyzing the thermal sensitivity of PEDF and the effects of protein concentration and pH are shown in Figure 2(CF). The absorbance of protein solutions (350 nm) was measured at a heating rate of 1°C / min from 10°C to 70°C, and the turbidity profile was analyzed. The transition temperature (Tt) at each condition was defined as the inflection point of the absorbance profile.

[0181] PEDF-E in 10 mM PBS in Figure 2(C, D) 6-6-6 -The Tt of PEDF was measured as 32.44, 30.47, 29.27, and 28.27°C at concentrations of 10, 25, 50, and 100 μM, respectively. In addition, 25 μM PEDF-E 6-6-6 -The Tt of PEDF according to pH change was confirmed to be 34.07, 32.32, 30.42, and 28.52°C at pH 6.4, 6.8, 7.4, and 8.0, respectively.

[0182] PEDF-E in 10 mM PBS in Fig. 2(E, F) 12-6-12 -The Tt of PEDF was 23.17, 21.17, 20.37, and 19.48°C at concentrations of 10, 25, 50, and 100 μM, respectively. In addition, 25 μM PEDF-E 12-6-12 -Tt according to the pH change of PEDF was measured as 25.06, 23.21, 20.37, and 20.46°C at pH 6.4, 6.8, 7.4, and 8.0, respectively.

[0183] In both libraries, Tt tended to decrease as protein concentration and pH increased, which was consistent with previous reports.

[0184]

[0185] PEDF-E n-6-n -Thermal sensitivity of PEDF and the effect of ZnCl₂

[0186] PEDF-E n-6-n -The results of analyzing the thermal sensitivity of PEDF and the effect of ZnCl₂ are shown in Fig. 3. The absorbance in a protein solution (350 nm) was measured at a heating rate of 1°C / min from 10°C to 60°C, and the turbidity profile was analyzed. Tt under each condition was defined as the inflection point of the absorbance profile.

[0187] 10 μM concentration E in 10 mM HEPES in Figure 3(A) 6-6-6 , E 12-6-12 , PEDF-E 6-6-6 -PEDF and PEDF-E 12-6-12 -The Tt of PEDF was measured as 41.62, 31.22, 43.12, and 35.12°C, respectively.

[0188] E 12-6-12 and PEDF-E 12-6-12 -PEDF's Tt is E each 6-6-6 and PEDF-E 6-6-6 -10.04°C and 7.90°C lower than PEDF, which is consistent with previous reports that Tt decreases as the length of the EBP block increases. PEDF-E6-6-6 -PEDF and PEDF-E 12-6-12 -PEDF's Tt is E 6-6-6 and E 12-6-12 The values ​​were 1.50°C and 4.00°C higher, respectively, which is presumed to be because the charge of the PEDF 34-mer hindered the aggregation of the adjacent hydrophobic EBP block.

[0189] In Figure 3(B), PEDF-E 12-6-12 -The effect of ZnCl₂ on the thermal sensitivity of PEDF was analyzed. Since this protein has a transition temperature below 37°C, E with a Tt above 40°C 6-6-6 and PEDF-E 6-6-6 -Additional experiments were conducted compared to PEDF.

[0190] In theory, a single E 12-6-12 and PEDF-E 12-6-12 -PEDF contains 720 histidines (His), and therefore [His]:[Zn] for 10 μM protein 2+ ] The ratios were calculated to be 16:1, 8:1, and 4:1 at 45, 90, and 180 μM ZnCl₂, respectively.

[0191] 10 μM PEDF-E in Figure 3(B) 12-6-12 -The Tt of PEDF decreased from 35.22°C in the absence of ZnCl₂ to 32.42°C and 29.62°C at 45 and 90 μM ZnCl₂, respectively. In the presence of 180 μM ZnCl₂, the protein aggregated at 20°C and no phase transition was observed.

[0192] The results that the Tt of EBP containing histidine decreased as the concentration of ZnCl₂ increased, and the phenomenon of lower critical solution temperature (LCST) was not observed above a certain concentration were consistent with previous studies. The histidine-rich EBP block was Zn 2+ It is converted into a multimer through chelation, and Tt is reduced accordingly.

[0193]

[0194] PEDF-E 12-6-12 -Thermo-induced self-assembly of PEDF and the effect of ZnCl₂

[0195] PEDF-E according to changes in ZnCl₂ concentration 12-6-12 -To analyze the self-assembly of PEDF, the hydration radius (R) below and above the transition temperature at various ZnCl₂ concentrations H ) was measured.

[0196] 10 μM PEDF-E in Figure 3(C, D) 12-6-12 -R of PEDF H was a soluble monomer of 7.85 ± 0.65 nm in the absence of ZnCl₂ at 20°C, which increased to 46.55 ± 5.89 and 62.60 ± 7.66 nm upon addition of 45 and 90 μM ZnCl₂, respectively. When 180 μM ZnCl₂ was added, PEDF-E 12-6-12 -PEDF was aggregated into particles larger than 1 μm in size.

[0197]

[0198] PEDF-E with and without ZnCl₂ 12-6-12 -Self-assembly and structural analysis of PEDF

[0199] In the absence of ZnCl₂, PEDF-E 12-6-12 -PEDF existed as a soluble monomer with a radius of less than 10 nm at 20°C, and formed nanostructures with a radius of more than 1000 nm through self-assembly at 37°C.

[0200] In the presence of ZnCl₂, it remained soluble even at 20°C, but the histidine-rich EBP block was Zn 2+ Interacts with the intermolecular His-Zn 2+ A bond is formed, and thus the hydration radius (R H ) increased to less than 100 nm. At 37°C, Zn 2+Histidine-rich EBPs, including those containing Zn, became hydrophobic and consequently 2+ The self-assembled multivalent structure formed by combining these hydrophobic blocks is Zn 2+ Self-assembled into nanostructures of smaller size than those without.

[0201] PEDF-E with 45 or 90 μM ZnCl₂ added 12-6-12 -R of PEDF H was significantly reduced to less than 500 nm compared to the case without ZnCl₂. In general, the radius of the self-assembled structure increases as the volume fraction of lipids increases. The volume of the hydrophilic EBP block remains constant at 20°C and 37°C regardless of the presence of ZnCl₂, but in the presence of Zn 2+ The hydrophobic core containing Zn 2+ It is likely to be packed more densely than without it. Therefore, Zn 2+ PEDF-E included 12-6-12 -The volume fraction of the hydrophobic core of PEDF is Zn 2+ This is lower than in the absence of ZnCl₂, and consequently, self-assembles into smaller structures when ZnCl₂ is present.

[0202]

[0203] PEDF-E 12-6-12 -Self-assembled structures and imaging of PEDF

[0204] PEDF-E 12-6-12 -PEDF self-assembled structures were prepared in deionized water (DW) and PBS and imaged. PEDF-E 12-6-12 -PEDF was completely dissolved in 4 M urea solution to increase the transition temperature (Tt), and then dialyzed into DW or PBS for self-assembly.

[0205] As a result, PEDF-E 12-6-12-PEDF self-assembled into fully filled nanostructures or toroidal structures with radii of 2700 nm or 2400 nm, respectively, as shown in Figures 4(AD) and 4(EH).

[0206] PEDF-E for additional in vitro HUVEC antiangiogenic experiments and imaging 12-6-12 -The ratio of PEDF and ZnCl₂ was set to 1:9.

[0207] PEDF-E with and without ZnCl₂ 12-6-12 -Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) imaging were performed to confirm the PEDF self-assembled structure.

[0208] The swollen state imaging using CLSM in the presence or absence of ZnCl₂ was performed at 37°C in 10 mM HEPES, and the results are shown in Fig. 4(I, J), respectively. In the absence of ZnCl₂ (Fig. 4(I)), the structures showed uniform spherical structures of less than 2 μm, which was comparable to the PEDF-E at 37°C in the absence of ZnCl₂. 12-6-12 -The results were consistent with the DLS results showing that the PEDF self-assembled into nanostructures with a radius of 1445.33 ± 82.04 nm.

[0209] TEM imaging (Fig. 4(K, L)) showed 10 μM PEDF-E in 10 mM HEPES containing 90 μM ZnCl₂ 12-6-12 -PEDF was prepared by drying at 37°C. The spherical nanostructures observed in the TEM images showed fully filled nanostructures with a radius of less than 200 nm, which was smaller than the hydrated radius (221.73 ± 58.33 nm) measured by DLS due to the drying process.

[0210] Zn 2+Due to the complexation with , protein nanostructures could be observed even without phosphotungstic acid (PTA) staining.

[0211]

[0212] PEDF-E 12-6-12 - Inhibitory effect of PEDF self-assembled nanostructures on HUVEC tube formation

[0213] PEDF-E depending on whether ZnCl₂ is added 12-6-12 -The inhibitory effect of the PEDF self-assembly structure on tube formation in HUVECs was further evaluated in vitro. The results are shown in Fig. 5.

[0214] PEDF-E in the range of 0.01 - 1000 nM 12-6-12 - PEDF (with or without ZnCl₂) was treated on HUVECs, and migration and tube formation were induced in HUVECs by rhVEGF165 (50 ng / mL). 0.01 and 1000 nM PEDF-E 12-6-12 -PEDF (with or without ZnCl₂) was treated together with rhVEGF165 and used as a control group.

[0215] After treating calcein-AM-stained HUVECs for 4 hours, fluorescence microscopic images of the cells were taken, and the results are presented in Fig. 5(A). In addition, a graph showing the degree of tube formation based on the normalized tube length of HUVECs is shown in Fig. 5(B).

[0216] As shown in Fig. 5(A), HUVECs treated with rhVEGF165 migrated more actively than HUVECs not treated with rhVEGF165 and formed a tube-shaped network.

[0217] The tube lengths of migrated HUVECs were set to 0% and 100%, respectively, depending on whether rhVEGF165 was treated or not, and the migrated tube lengths of HUVECs treated with and without ZnCl₂ were normalized.

[0218] E with or without ZnCl₂ under rhVEGF165 12-6-12 , E 12-6-12 + The degree of tube formation in HUVECs treated with ZnCl₂ did not show a significant difference from the group treated with rhVEGF165 alone, regardless of concentration.

[0219] However, PEDF-E with or without ZnCl₂ under VEGF stimulation 12-6-12 -Tube formation in the PEDF treatment group showed a concentration-dependent decrease in the range of 10 to 1000 nM.

[0220] In the absence of ZnCl₂, PEDF-E 12-6-12 -PEDF inhibited tube formation in HUVECs at concentrations above 10 nM, and the inhibitory effect on HUVECs treated with the protein at 1000 nM showed lower tube formation than that of HUVECs without VEGF stimulation.

[0221] When containing ZnCl₂, PEDF-E 12-6-12 -PEDF inhibited tube formation in HUVECs at 0.01 nM, and at concentrations higher than 100 nM of the protein, the tube formation rate was lower than that of HUVECs without VEGF stimulation.

[0222] When containing ZnCl₂, PEDF-E 12-6-12 -PEDF inhibited tube formation in HUVECs at lower concentrations than when no ZnCl₂ was added. Furthermore, PEDF-E containing ZnCl₂ 12-6-12 -PEDF showed a higher inhibitory effect than when ZnCl₂ was not added at that concentration.

[0223] Additionally, 1000 nM PEDF-E without ZnCl₂ 12-6-12 -HUVECs treated with PEDF showed lower tube formation than those treated with ZnCl₂ (see Figure 5(B)).

[0224]

[0225] PEDF-E 12-6-12- Tube formation inhibition effect according to the type of PEDF self-assembled nanostructure

[0226] PEDF-E 12-6-12 -The inhibitory effect of PEDF self-assembled nanostructures on HUVEC tube formation was further evaluated in vitro. The results are shown in Figs. 6 and 7.

[0227] PEDF-E at concentrations of 0.001 - 1000 nM in HEPES with or without ZnCl₂ 12-6-12 -PEDF formed vesicle structures or coacervate structures, and PEDF-E in the concentration range of 0.001 - 1000 nM in PBS 12-6-12 -PEDF toroidal structures were prepared. Migration and tube formation of HUVECs treated with each structure were stimulated by rhVEGF165 (50 ng / mL) in Matrigel.

[0228] Cell fluorescence microscopy images of HUVECs stained with Calcein-AM for 4 hours are presented in Figs. 6(A) and 7(A), a graph showing the degree of tube formation calculated based on the normalized tube length of HUVECs is presented in Fig. 6(B), and a graph showing the degree of tube formation based on the number of normalized junctions is presented in Fig. 7(B).

[0229] As shown in Figures 6(A) and 7(A), HUVECs treated with rhVEGF165 migrated more and formed networks than HUVECs not treated with rhVEGF165.

[0230] The tube length or number of junction points of migrated HUVECs in the presence or absence of rhVEGF165 was set to 0 or 100%, respectively, and the tube length or number of junction points of migrated HUVECs treated with PEDF-E12-6-12-PEDF was normalized.

[0231] PEDF-E in the presence or absence of ZnCl2 under rhVEGF165 stimulation 12-6-12Tube formation in HUVECs cultured with PEDF was dose-dependently reduced in the range of 0.001–1000 nM. PEDF-E in the absence or presence of ZnCl2 12-6-12 -PEDF inhibited tube formation in HUVECs at concentrations of 1 and 0.001 nM or higher, respectively, and the extent of protein-treated HUVECs at 1000 nM was lower than that of HUVECs without rhVEGF165 stimulation, regardless of ZnCl2. The concentration range and extent of inhibition were consistent with the results in Figure 5. PEDF-E in PBS 12-6-12 -The toroidal structure of PEDF inhibited tube formation in HUVECs in a dose-dependent manner at 0.001 nM and completely suppressed rhVEGF165 stimulation, resulting in a lower degree of tube formation than HUVECs not treated with rhVEGF165.

[0232] The degree and pattern of inhibition of toroidal structures were similar to those of vesicle structures without ZnCl2, which was probably due to the size of PEDF-E without ZnCl2 in HEPES and PBS. 12-6-12 -This may be because the respective radii of PEDF with PEDF and ZnCl2 were 1,445.33, 221.73, and 2,400 nm.

[0233]

[0234] PEDF-E 12-6-12 -Cytotoxicity of PEDF structures

[0235] Considering the mechanism of PEDF 34-mer for antiangiogenic activity related to cell viability, PEDF-E in the presence or absence of ZnCl2 12-6-12 -The cytotoxicity of the PEDF structure was quantified using the WST-8 reagent and the HUVEC viability was shown in Figure 8.

[0236] PEDF 34-mer has been reported to inhibit ATP synthase, thereby reducing cell viability and participating in several cell death processes.

[0237] PEDF-E in the range of 0.01–1000 nM depending on the presence or absence of ZnCl2 12-6-12 - PEDF was treated on HUVECs and cultured for 4 and 12 hours to determine the effect on viability while inhibiting HUVEC tube formation. E depending on the presence or absence of ZnCl2 12-6-12 was treated with HUVEC as a control. The viability of HUVEC cultured in the medium was set to 100%, and other values ​​were normalized.

[0238] ZnCl2 and E 12-6-12 HUVECs treated with PEDF-E did not show a significant decrease in viability compared to HUVECs cultured in medium, regardless of the concentration. PEDF-E with or without ZnCl2 12-6-12 -The viability of HUVECs treated with PEDF did not decrease significantly regardless of the concentration. This is because PEDF-E 12-6-12 -The structure of PEDF indicates that it is not cytotoxic to HUVECs and inhibits HUVEC migration and tube formation without reducing cell viability.

[0239]

[0240] conclusion

[0241] A genetically engineered PEDF 34-mer and histidine-containing EBP triblock copolypeptide (PEDF-EBP) was expressed in bacteria, purified via a chromatography-free purification method, and self-assembled into nanostructures containing the multivalent antiangiogenic peptide, PEDF 34-mer.

[0242] Changes in self-assembled structures and buffer solutions and histidine-Zn 2+ The size change due to ignition is the hydration radius (R H ) was confirmed through measurement and imaging.

[0243] PEDF-EBP existed as a soluble monomer below the transition temperature (Tt) and self-assembled into nanostructures larger than 2 μm above Tt. In the presence of ZnCl₂, the size of the nanostructures decreased to less than 1 μm.

[0244] The formation and size reduction of nanostructures were clearly confirmed through DLS measurements and TEM images. Structures larger than 2 μm in the absence of ZnCl₂ and structures smaller than 1 μm in the presence of ZnCl₂ were identified as vesicular structures and coacervates, respectively, through image analysis.

[0245] Both constructs >2 μm in diameter without ZnCl₂ and constructs <1 μm in diameter with ZnCl₂ inhibited tube formation in HUVECs in a concentration-dependent manner under VEGF stimulation. Furthermore, both constructs inhibited tube formation as much as the condition without rhVEGF165 under VEGF stimulation.

[0246] In particular, coacervate containing ZnCl₂ showed a higher inhibitory effect on HUVEC tube formation than vesicular structures without ZnCl₂.

[0247] That is, the self-assembled structure formed by the multi-stimulus responsive block copolypeptide of the present invention can be utilized as an improved anti-angiogenic therapeutic agent with a multivalent nanostructure whose size can be controlled.

[0248]

[0249] While specific portions of the contents of this application have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and do not limit the scope of this application. Therefore, the substantial scope of this application is defined by the appended claims and their equivalents.

Claims

1. A multi-block copolypeptide comprising a peptide derived from epithelial-derived factor (PEDF) and an elastin-based polypeptide (EBP), A multi-block copolypeptide represented by the following formula 1. [Formula 1] [A]-[B]n-[C]m-[B]n-[A] In the above equation 1, The above A block is a PDEF-derived peptide, The above B block or C block are different EBPs, n and m are integers that are equal or different and represent the number of times each block is repeated.

2. In paragraph 1, The above B block is a multi-block copolypeptide, which is a hydrophobic EBP.

3. In paragraph 2, The above hydrophobic EBP is a multi-block copolypeptide comprising blocks consisting of an amino acid sequence of SEQ ID NO: 1 in a continuous and repetitive manner.

4. In paragraph 3, A multi-block copolypeptide wherein the number of repetitions of the hydrophobic EBP block is 1 to 36.

5. In paragraph 1, The above C block is a multi-block copolypeptide, which is a hydrophilic EBP.

6. In paragraph 5, The above hydrophilic EBP is a multi-block copolypeptide comprising blocks consisting of an amino acid sequence of SEQ ID NO: 2 in a continuous and repetitive manner.

7. In paragraph 6, A multi-block copolypeptide wherein the number of repetitions of the hydrophilic EBP block is 1 to 36.

8. In paragraph 1, The above PEDF-derived peptide is a multi-block copolypeptide comprising the amino acid sequence of SEQ ID NO:

3.

9. A multi-stimulus-responsive drug delivery system comprising the multi-block copolypeptide of claim 1 and exhibiting stimuli-responsiveness.

10. In paragraph 9, A multi-stimulus responsive drug delivery system, characterized in that the above stimulus is at least one of temperature, protein concentration, pH, or Zn ion.

11. In paragraph 9, A multi-stimulus responsive drug delivery system wherein the copolypeptide forms a nanostructure whose size is controlled by the stimulus.

12. In paragraph 9, The above nanostructure is a multi-stimulus responsive drug delivery system having a vesicle, coacervate or toroidal structure.

13. A pharmaceutical composition comprising a multi-block copolypeptide comprising a peptide derived from epithelial-derived factor (PEDF) and an elastin-based polypeptide (EBP), A pharmaceutical composition represented by the following formula 1. [Formula 1] [A]-[B]n-[C]m-[B]n-[A] In the above equation 1, The above A block is a PDEF-derived peptide, The above B block is a hydrophobic EBP, The above C block is a hydrophilic EBP, n and m are integers that are equal or different and represent the number of times each block is repeated.

14. In paragraph 13, A pharmaceutical composition wherein the hydrophobic EBP comprises a block consisting of an amino acid sequence of sequence number 1 in a continuous and repetitive manner.

15. In paragraph 13, A pharmaceutical composition wherein the hydrophilic EBP comprises a block consisting of an amino acid sequence of sequence number 2 in a continuous and repetitive manner.

16. In paragraph 13, A pharmaceutical composition, wherein the number of repetitions of the hydrophilic EBP block is 1 to 36.

17. In paragraph 13, A pharmaceutical composition, wherein the PEDF-derived peptide comprises an amino acid sequence of SEQ ID NO:

3.

18. A pharmaceutical composition for inhibiting angiogenesis, characterized in that it prevents or treats a disease caused by angiogenesis, comprising the pharmaceutical composition of Article 13.

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