Self-assembled nanovesicle structure based on fusion polypeptide for inhibiting angiogenesis and its use

Abstract

The present invention relates to a polypeptide-based self-assembled nanovesicle structure for inhibiting neovascularization. The polypeptide-based nanovesicle structure for inhibiting neovascularization according to the present invention can be used to treat neovascularization-related diseases.

Description

Technology Field

[0001] The present invention relates to a fusion polypeptide-based nanovesicle structure for inhibiting neovascularization and its use. Background Technology

[0003] Nature-inspired spontaneous self-assemblies provide an understanding of the fundamental mechanisms and dynamics regarding the formation of high-dimensional structures and thermodynamically preferred nanostructures, enabling the development of biomimetic smart biomaterials in the biomedical field. Protein-based soft biomaterials have emerged as highly important materials due to their unique properties. Advancements in molecular biology and protein engineering over the past few decades have enabled a deep understanding of the relationships between protein sequences, structures, and functions. Peptide-based self-assembling nanostructures are attractive due to their biocompatibility with physiological conditions, such as biodegradability and the regulation of sequence-structure-function at the genetic level. Consequently, there is tremendous effort to develop and utilize biomimetic soft materials that mimic the behavior of their natural counterparts. Protein engineering approaches for the development of customized protein-based biopolymers generally focus on structural repeats or functional domains, utilizing these domains to design and develop multifunctional biomaterials such as elastin, silk, and collagen-based materials. In particular, stimulus-responsive materials are critical due to their ability to exhibit desirable and localized assembly and disassembly in response to a wide range of stimuli.

[0004] The development of nanocarriers as drug delivery systems has increased over the past few decades. Various synthesis methods and starting materials have been used for the development of nanoparticles. The ability to possess excellent colloidal stability in blood, along with the potential to load various small molecules such as drugs or imaging agents, is attractive to biotechnologists. While passive targeting of tumor cells through enhanced permeability and retention effects (EPR)—utilizing nanoparticle geometry confined to the nanoscale to penetrate tumor cells—has garnered significant interest, passive targeting does not directly target tumor cells but relies on the local accumulation of nanoparticles within the tumor. The fact that tumor cells express numerous surface proteins highly specific to particular cell types implies that these surface proteins can be used as targets for the direct targeting of tumor cells. In these efforts, Chilkoti and colleagues developed polypeptide-based self-assembling micelles functionalized with tumor cell targeting peptides for tumor cell targeting (Non-patent Literature 1 and Non-patent Literature 2). The ligand valence of the nanoparticles is very important as it determines the interaction between the tumor cell and the target peptide. However, this interaction between the tumor cell and the micelle expressing the target peptide in the corona can be limited because the size of the micelle is relatively small.

[0005] In contrast, vesicle-like self-assembled nanostructures are very common in living organisms and can encapsulate and transport small molecule payloads by remaining isolated from the external environment. This has led to increased interest in the development of vesicles for drug delivery applications. Currently, phospholipid-based polymerosomes and macromolecular surfactant vesicles are used as drug delivery carriers, but these macromolecules are very difficult to functionalize and often have poor biocompatibility.

[0006] Angiogenesis, an essential and fundamental biological process, plays a crucial role in blood vessel development and is primarily regulated by vascular endothelial growth factor (VEGF). Disruption of the strictly regulated balance between positive and negative regulators of physiological angiogenesis is associated with tumor angiogenesis. Tyrosine kinases, which are vascular endothelial growth factor receptors, are primarily expressed in endothelial cells and are important for angiogenesis and vasculogenesis. Vascular endothelial growth factor (VEGF) is involved in downstream signaling and many other responses necessary for angiogenesis by selectively binding to vascular endothelial growth factor receptor 1 (VEGFR1, or fms-like tyrosine kinase-1 (FLT1)) and vascular endothelial growth factor receptor 2 (VEGFR2). Numerous strategies have been proposed, including the development of antagonists such as anti-VEGFR1 monoclonal antibodies for site-specific targeting of vascular endothelial growth factor receptors in abnormal angiogenesis.

[0007] Recently, tremendous effort has been concentrated on the development of anti-FLT1 (Gly-Asn-Gln-Trp-Phe-Ile), a site-specific and selective hexapeptide, using a high-throughput screening system from a positional scanning synthetic peptide combinatorial library. Anti-FLT1 competitively inhibits the binding of many other growth factors, including placental growth factor (PIGF) as well as endothelial growth factor (VEGF), to FLT1. Several studies have shown that anti-FLT1 is a potential therapeutic candidate for various neovascular diseases such as cancer, retinal ischemia, diabetic retinopathy, and arthritis due to its ability to inhibit endothelial cell migration and morphogenesis.

[0008] Recently, self-assembled micelle nanostructures have been fabricated by conjugating hyaluronic acid (HA) with anti-FLT1 for the treatment of corneal angiogenesis, bioimaging, and lung applications. However, there are problems with the polydispersity of the nanostructures, along with reduced and inconsistent conjugation of HA.

[0009] To solve the above problem, the inventors continued their research and developed a self-assembling vesicle based on elastin and resilin-based diblock (diblock) copolypeptide, thereby completing the present invention. Prior art literature

[0011] 1: Dzuricky, M.; Xiong, S.; Weber, P.; Chilkoti, AJN l., Avidity and cell uptake of integrin-targeting polypeptide micelles is strongly shape-dependent. 2019, 19 (9), 6124-6132.; 26.2: Weber, P.; Dzuricky, M.; Min, J.; Jenkins, I.; Chilkoti, AJB, Concentration-Independent Multivalent Targeting of Cancer Cells by Genetically Encoded Core-Crosslinked Elastin / Resilin-like Polypeptide Micelles. 2021, 22 (10), 4347-4356.) The problem to be solved

[0012] Accordingly, the present invention aims to provide a polypeptide-based self-assembled nanovesicle structure for inhibiting neovascularization.

[0013] In addition, the present invention has another objective of providing a composition for treating diseases caused by neovascularization comprising the self-assembled nanovesicle structure.

[0014] In addition, the present invention has another objective of providing a theranostic nanoprobe for a disease caused by neovascularization comprising the self-assembled nanovesicle structure.

[0016] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0018] To achieve the above-mentioned objectives of the present invention, the present invention provides the following fusion polypeptide-based self-assembled nanovesicle structure for inhibiting neovascularization:

[0019] A fusion polypeptide for inhibiting angiogenesis, comprising an anti-FLT1 peptide represented by SEQ ID NO. 1; an elastin-based polypeptide (EBP) represented by SEQ ID NO. 2 linked to the peptide; and a resilin-based polypeptide (RBP) represented by SEQ ID NO. 3 linked to the EBP.

[0020] It is formed by self-assembling into a double layer nanovesicle of an anti-FLT1 layer and an EBP-RBP layer at a temperature below the lower critical solution temperature (LCST) of the EBP and below the upper critical solution temperature (UCST) of the RBP.

[0021] At temperatures below the LCST of the EBP and below the UCST of the RBP, the anti-FLT1 forms a surface layer, the RBP aggregates, and the EBP becomes soluble, allowing it to self-assemble into a double-layer nanovesicle of the anti-FLT1 layer and the EBP-RBP layer.

[0022] The temperatures below the LCST of the above EBP and below the UCST of the above RBP may be within the body temperature range.

[0023] The above nanovesicle structure may have a multivalent anti-FLT1 peptide.

[0024] In another aspect, the present invention provides a composition for treating a disease caused by neovascularization comprising the self-assembled nanovesicle structure.

[0025] In another aspect, the present invention provides a theranostic nanoprobe for a disease caused by neovascularization, comprising: a fluorescent dye; and the self-assembled nanovesicle structure.

[0026] The above-mentioned diseases caused by neovascularization may be one or more selected from diabetic retinopathy, retinopathy of prematurity, macular degeneration, choroidal neovascularization, neovascular glaucoma, ocular disease caused by corneal neovascularization, rejection reaction during corneal transplantation, corneal edema, corneal opacity, cancer, hemangioma, angiofibroma, rheumatoid arthritis, and psoriasis. Effects of the invention

[0028] The polypeptide-based nanovesicle structure for inhibiting neovascularization according to the present invention provides an advanced drug delivery system for treating neovascularization-related diseases such as retinal, corneal, and choroidal angiogenesis, tumor growth, cancer cell metastasis, diabetic retinopathy, and asthma. Brief explanation of the drawing

[0030] Figure 1 is a schematic diagram showing the molecular design, self-assembly, vascular endothelial growth factor receptor (VEGFR1) targeting, and intracellular localization of E6R6 (control) and AF1-E6R6. (A) Amino acid sequences of anti-Flt1 (AF1), elastin-like polypeptide (E1), and resilin-like polypeptide used in the molecular design of AF1-E6R6, and a schematic diagram of the thermally induced self-assembled bilayer vesicle structure of AF1-E6R6. Aggregation of the R6 block below UCST and the soluble E6 block below LCST self-assembles into a thermodynamically preferred vesicle, as can be seen in the CLSM vesicle image. (B) A schematic diagram of AF1-E6R6-based vesicles for in vitro targeting of vascular endothelial growth factor receptor 1 (VEGFR1). (C) As shown by tubing analysis performed on HUVECs of AF1-E6R6, AF1-E6R6-based vesicles express an anti-Flt1 peptide on their surface for site-specific targeting of vascular endothelial growth factor receptor 1 (VEGFR1) in the presence of other ligands, including vascular endothelial growth factor (VEGF). AF1-E6R6-based vesicles inhibit tubular formation in HUVECs. (D) shows the cellular localization of AF1-E6R6 vesicles compared to E6R6, which does not exhibit cell adhesion. Figure 2 shows the gene sequencing, synthesis, purification, and characterization of the E6R6 and AF1-E6R6 blocks. In the first step, pET21(+) was modified for RDL, and E and R monoblocks were synthesized and multiplexed. Genes for the fusion polypeptide of the AF1-E6R6 block copolypeptide were generated using an AF1 oligonucleotide cassette and a plasmid containing E6R6 with recognition sites for BseRI, XbaI, AcuI, and BamHI. After digesting the gene containing the first E6R6 plasmid (pET21-a) with BseRI, an anti-Flt1 peptide was inserted. (Ai) 1% agarose gel images of E6R6 and AF1-E6R6 block genes after restriction with XbaI and BamHI, Lane 1) DNA ladder, Lane 2) E6R6 gene, Lane 3) AF1-E6R6 gene. (A-ii) SDS-PAGE images of E6R6 and AF1-E6R6 blocks after purification by ITC. The gradient gel (4-20%) was visualized by copper staining. Expected molecular weights are indicated under the gel bands. (A-iii) Purification of E6R6 and AF1-E6R6 blocks via size exclusion chromatography (SEC). Absorbance was measured at 280 nm. (B) Concentration-dependent thermal profiles of E6R6 and AF1-E6R6 blocks in PBS from 10°C to 90°C at a heating rate of 1°C / min. (C) Thermal profile of E6R6 and AF1-E6R6 blocks at a heating rate of 0.5°C per minute. (D) pH-dependent thermal profiles of the E6R6 and AF1-E6R6 blocks at a heating rate of 1°C / min. Both samples were prepared in phosphate buffer at pH 6.4 (weakly acidic), 7.4 (physiologically relevant), and 8.4 (weakly basic). (E) Salt-dependent thermal profiles of the E6R6 and AF1-E6R6 blocks at a heating rate of 1°C / min. The concentration of NaCl ranged from 0.01M to 0.1M, and in all thermal profiling experiments, the samples were equilibrated for 10 minutes at the starting temperature. Figure 3 shows transmission electron microscopy (TEM) images to confirm the morphology of the nanostructures and statistical analysis of the E6R6 (control) and AF1-E6R6 blocks. (Ai), (A-ii) and (Bi), (B-ii) are TEM images of the E6R6 and AF1-E6R6 blocks, respectively. TEM samples were prepared at 37°C, and 2% (w / v) tungstic acid was used as a negative stain. (C) shows the statistical analysis of the vesicle size distribution in TEM for E6R6 and AF1-E6R6. In TEM, the average diameter of E6R6-based vesicles is 281.8 nm, while the average diameter of AF1-E6R6-based vesicles is 233.3 nm. The scale bar in (Ai) and (Bi) is 1000 nm. The scale bars of (A-ii) and (B-ii) are 500 nm. Figure 4 shows the hydrodynamic radii, confocal laser scanning microscope (CLSM) images, and statistical analysis results for E6R6 and AF1-E6R6 blocks at 25 μM to confirm self-assembly into vesicle structures in the swollen state. (A) Hydrodynamic radii of E6R6 and AF1-E6R6 blocks at 37°C. Samples were prepared in PBS at a concentration of 25 μM and equilibrated at 37°C for 10 minutes prior to measurement. (B) Photographic images of E6R6 and AF1-E6R6 blocks in PBS at 37°C. Compared to PBS, both E6R6 and AF1-E6R6 blocks exhibited slight turbidity while maintaining a colloidal state, serving as an indicator of vesicle structures. (C) is a brightfield image of E6R6 and AF1-E6R6 blocks, respectively. (D) is a fluorescence image of AF1-E6R6 vesicles labeled with FITC. For all microscopic experiments, samples were incubated at 37°C prior to imaging. All images were taken at 37°C. (E) shows the statistical analysis of the vesicle size distribution for E6R6 and AF1-E6R6 in the swollen state. The average diameter of E6R6-based vesicles is 2.84 μm, while the average diameter of AF1-E6R6-based vesicles is 2.01 μm. Figure 5 shows the internalization of E6R6 and AF1-E6R6-based vesicles. Each block was conjugated with FITC before analysis. (A) is a schematic diagram of the internalization of AF1-E6R6-based vesicles in the presence of anti-Flt1. (Bi to vi) show the internalization of AF1-E6R6-based vesicles at 1 μM after 4 hours of culture, whereas (C-i to vi) E6R6-based vesicles do not bind to cells and thus cannot be internalized. Figure 6 shows the results of the cytotoxicity analysis of E6R6 and AF1-E6R6 blocks before and after FITC conjugation. . (a) and (b) the viability of E6R6 and AF1-E6R6 blocks without or containing FITC conjugation at concentrations of 0.1 μM, 0.5 μM, and 1 μM for E6R6 blocks, and 0.1 μM, 0.25 μM, 0.5 μM, 0.75 μM, and 1 μM for AF1-E6R6 blocks, respectively. Figure 7 shows the results of in vitro tubing analysis of E6R6 and AF1-E6R6 blocks. (a) Fluorescence microscopy image of calcein-AM-labeled HUVEC and (b) degree of tube formation inhibition. am The degree of inhibition was quantified from the image in (a). Calcein-AM labeled HUVEC (2Х10 4 Canine cells (well) were plated on Matrigel-pre-coated 48-well plates and treated with VEGF and various concentrations of E6R6 and AF1-E6R6 blocks for 4 hours. Specific details for implementing the invention

[0031] In one aspect, the present invention provides the following fusion polypeptide-based self-assembled nanovesicle structure for inhibiting neovascularization:

[0032] A fusion polypeptide for inhibiting angiogenesis, comprising an anti-FLT1 peptide represented by SEQ ID NO. 1; an elastin-based polypeptide (EBP) represented by SEQ ID NO. 2 linked to the peptide; and a resilin-based polypeptide (RBP) represented by SEQ ID NO. 3 linked to the EBP.

[0033] It is formed by self-assembling into a double layer nanovesicle of an anti-FLT1 layer and an EBP-RBP layer at a temperature below the lower critical solution temperature (LCST) of the EBP and below the upper critical solution temperature (UCST) of the RBP.

[0034] At temperatures below the LCST of the EBP and below the UCST of the RBP, the anti-FLT1 forms a surface layer, the RBP aggregates, and the EBP becomes soluble, allowing it to self-assemble into a double-layer nanovesicle of the anti-FLT1 layer and the EBP-RBP layer.

[0035] The above temperatures below the LCST of EBP and below the UCST of RBP may be within the body temperature range, which means 36 to 37.5℃.

[0036] The above nanovesicle structure may have a multivalent anti-FLT1 peptide.

[0037] In another aspect, the present invention provides a composition for treating a disease caused by neovascularization comprising the self-assembled nanovesicle structure.

[0038] In another aspect, the present invention provides a theranostic nanoprobe for a disease caused by neovascularization, comprising: a fluorescent dye; and the self-assembled nanovesicle structure.

[0039] The above-mentioned diseases caused by neovascularization may be one or more selected from diabetic retinopathy, retinopathy of prematurity, macular degeneration, choroidal neovascularization, neovascular glaucoma, ocular disease caused by corneal neovascularization, rejection reaction during corneal transplantation, corneal edema, corneal opacity, cancer, hemangioma, angiofibroma, rheumatoid arthritis, and psoriasis.

[0041] The term "anti-angiogenic peptide" used in the present invention refers to a peptide that inhibits the formation of new blood vessels. As a specific example, the anti-Flt1 peptide specifically binds to Flt1, a vascular endothelial growth factor (VEGF) receptor, and inhibits the formation of new blood vessels.

[0042] The fusion polypeptide of the present invention acts to inhibit the formation of new blood vessels.

[0043] The term "amino acid" used in the present invention means natural amino acids or artificial amino acids, preferably natural amino acids. For example, the amino acids include glycine, alanine, serine, valine, leucine, isoleucine, methionine, glutamine, asparagine, cysteine, histidine, phenylalanine, arginine, tyrosine, or tryptophan.

[0044] The properties of the above 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.

[0045] The abbreviations Gly (G), Ala (A), etc. used in this specification are abbreviations for amino acids. Gly is an abbreviation for glycine, and Ala is an abbreviation for alanine. Additionally, glycine is also referred to as G, and alanine as A. The above abbreviations are expressions widely used in this technical field.

[0046] As used herein, the term "polypeptide" refers to any polymeric chain of amino acids. The terms "peptide" and "protein" may be used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" includes natural or synthetic proteins, protein fragments, and polypeptide analogs of protein sequences. A polypeptide may be a monomer or a polymer.

[0047] The "elastin-based polypeptides (EBPs)" of the present invention are also referred to as "elastin-like polypeptides (ELPs)." This is a term widely used in the technical field of the present invention.

[0048] In this specification, the above Xaa (or X) referred to as "guest residue." The above X aa Various types of EBP according to the present invention can be manufactured by introducing various types.

[0049] Elastin is a major protein component of the extracellular matrix (ECM) and consists of an elastomeric domain and a crosslinking domain. The elastomeric domain is composed of hydrophobic amino acids and repetitive peptides such as VPGG, VPGVG, and APGVGV. Elastin-based polypeptides (EBPs) are thermally responsive biomolecules derived from the elastomeric domain. Elastin is a major protein component in the extracellular matrix (ECM). EBPs are modified to possess thermal sensitivity based on the elastomeric domain and are composed of Val-Pro-(Gly or Ala)-X, a repeating unit of a pentapeptide, which is a peptide consisting of five amino acids. aa It possesses -Gly[VP(G or A)XG]. EBPs are heat-sensitive polypeptides, and their transition temperatures are easily controlled to form drug delivery nanostructures.

[0050] The above X aa is a guest residue and can be any amino acid except proline. EBPs are designed to be stimulus-responsive, biocompatible, biodegradable, and non-immunolytic based on elastomer domains. EBPs are heat-responsive biopolymers, consisting of many 'pentapeptide repeat units' such as Val-Pro-(Gly or Ala)-X aa It consists of -Gly, where X aaThe fourth amino acid in the repetitive unit mentioned above can be any amino acid other than Pro. EBPs exhibit lower critical solution temperature (LCST) behavior and show reversible phase transitions depending on temperature. Their LCST behavior has the advantage of enabling inverse transition cycling (ITC) based on protein purification methodologies that utilize the thermally induced phase transitions of EBPs. The ease of purification and stimulus-induced phase transitions of EBPs enable the genetic fusion of other functional proteins and peptides.

[0051] The EBP according to the present invention may be a polypeptide in which a pentapeptide is repeated, and this repeated polypeptide may form a polypeptide block (EBP block). Specifically, it may form a hydrophilic EBP block or a hydrophobic EBP block.

[0052] In the present invention, resilin is an elastic polymer protein present in the cuticle layer of insects and is known to provide various functions necessary for energy storage and repetitive movement. In the present invention, by modifying a portion of the above sequence, a resilin-based polypeptide (RBP) was prepared that maintains excellent mechanical properties while exhibiting phase transition behavior.

[0054] The amino acid sequences of anti-Flt1, EBP, and RBP used in the present invention are as shown in Table 1 below. EBP is denoted as E6 (Sequence No. 2) because [VPAAG VPAFG VPAAG VPAAGG VPAAG VPAFG] is repeated 6 times, and RBP is denoted as R6 (Sequence No. 3) because [GGRPSDSYGAPGGGN GGRPSSSYGAPGQGN] is repeated 6 times.

[0055] polypeptide order Sequence number Anti-Flt1 ( AF1 ) GNQWFI 1 EBP(E6) VPAAG VPAFG VPAAG VPAAGG VPAAG VPAFG VPAAG VPAFG VPAAG VPAAGG VPAAG VPAFGVPAAG VPAFG VPAAG VPAAGG VPAAG VPAFGVPAAG VPAFG VPAAG VPAAGG VPAAG VPAFGVPAAG VPAFG VPAAG VPAAGG VPAAG VPAFGVPAAG VPAFG VPAAG VPAAGG VPAAG VPAFG 2 RBP(R6) GGRPSDSYGAPGGGN GGRPSSSYGAPGQGNGGRPSDSYGAPGGGN GGRPSSSYGAPGQGNGGRPSDSYGAPGGGN GGRPSSSYGAPGQGNGGRPSDSYGAPGGGN GGRPSSSYGAPGQGNGGRPSDSYGAPGGGN GGRPSSSYGAPGQGNGGRPSDSYGAPGGGN GGRPSSSYGAPGQGN 3

[0056] The term "phase transition" refers to a change in the state of matter, such as water turning into steam or ice turning into water.

[0057] The above EBP is the transition temperature (transition temperature: T t They undergo a reversible phase transition at the lower critical solution temperature (LCST), also referred to as ). These, T t　 Water solubility is high below, but the temperature is T t If it exceeds, it becomes insoluble.

[0058] The above RBP undergoes a reversible phase transition at the upper critical solution temperature (UCST). They are highly insoluble below UCST, but become soluble above UCST.

[0059] The term "self-assembly" as used in the present invention refers to the formation of a specific structure in which individual components assemble themselves. For the purposes of the present invention, said self-assembly means forming a nanostructure in the form of a nanovesicle by forming a hydrophobic domain or core and a hydrophilic domain or shell, depending on the difference in solubilization under specific temperature conditions.

[0060] The term "nanovesicle" used in this invention refers to a small vesicle formed in the form of a nano-sized double membrane.

[0061] Therefore, the term "self-assembled nanovesicle" refers to a nano-sheathed vesicle structure formed by the self-assembly of its individual components.

[0062] The term "block copolypeptide" in the present invention refers to a polymer composed of two or more different polypeptide monomer blocks. In this specification, if composed of two different polypeptide monomer blocks, it is referred to as a "double-block (co)polypeptide," and if composed of three polypeptide monomer blocks, it is referred to as a "triple-block (co)polypeptide." Specifically, a polymeric copolypeptide is formed using EBP and RBP as monomers.

[0063] In the present invention, "theranostic nanoprobe" refers to a nano-sized probe capable of simultaneous therapy and diagnosis.

[0064] The term "nano" above includes a size range that is understood by people skilled in the art. Specifically, the size range may be from 0.1 to 1000 nm, more specifically from 10 to 1000 nm, more preferably from 20 to 500 nm, and even more preferably from 30 to 250 nm.

[0065] The present invention uses polypeptides as starting materials for the self-assembly of nanostructures and synthesizes vesicle-based self-assembled structures. The precise amino acid sequence for each block copolypeptide can generate an amphiphilic material capable of self-assembling into controlled nanostructures. Additionally, for the targeted delivery of these vesicles, various functional parts to confer function to the vesicles or stabilizing elements can be introduced into the sequence of the block copolypeptide.

[0067] To overcome the ligand valence associated with smaller nanoparticles, the inventors developed self-assembling vesicle structures based on elastin and regilin-based diblock copolypeptides. Elastin-based polypeptides (EBPs) have emerged as a new class of stimulus-responsive polymers possessing the functional characteristics of tropoelastin, a natural structural component of the extracellular matrix (ECM), and have inherited the self-assembly and mechanical properties of natural tropoelastin. Recombinant elastin-based peptides having a pentapeptide repeat of Val-Pro-Gly-Xaa-Gly (where Xaa can be any amino acid excluding proline) were developed using reversible temperature-responsive phase separation due to gradual changes in polypeptide structure and rapid changes in hydration properties with increasing temperature. Elastin-based polypeptides have become suitable candidates for biomedical applications due to the genetic modulation of stimulus responsiveness of elastin-based polymers based on various endogenous and exogenous factors, the feasibility of manipulating them into desired self-assembled nanostructures, and their biocompatibility. The multi-reactivity of EBPs can be utilized in various ways, such as allowing thermally reactive EBPs to convert into various structures at body temperature and to serve as nano-sized, confined reservoirs for drug release. These nanostructures can be surface-functionalized to act as functional epitopes or encapsulate small molecules within a core. Furthermore, elastin-based peptides are desirable candidates due to easy and inexpensive purification methods that utilize the thermal responsiveness of elastin-based peptides. The thermal responsiveness of elastin-based peptides can be refined by their low critical solution temperature (LCST), at which hydrophobic side chains are exposed at the protein-solvent interface, reducing the total surface area of ​​the polypeptide available for solvent exposure and causing phase separation of the elastin.By utilizing the LCST behavior of elastin-based polypeptides, recombinant EBPs can be purified using a non-chromatographic method called the reverse transition cycle (ITC). Genetic fusion of various functional peptides or polypeptides with elastin-based polymers can be used to develop multifunctional biomaterials for various biological applications. Elastin-fused polypeptides with excellent monodispersity and high yield can be biosynthesized via a cost-effective process using EBPs, which provide the ability to scale production.

[0068] Resilin-based polypeptides belong to a class of elastic proteins and are gaining significant importance due to properties including high mechanical strength, excellent elasticity, and genetic tunability. Natural resilins are found in specialized regions such as the thorax and tendons of many arthropods, such as *Drosophila melanogaster*, and possess various functions, including excellent elasticity and mechanical stability during high-speed repetitive movements. Recombinant resilins have been shown to respond to various stimuli, including temperature, pH, and light. The dual phase behavior (DBP) of recombinant resilins, which possesses both a lower critical solution temperature (LCST) and an upper critical solution temperature (UCST), makes them attractive candidates for the development of multi-reactive smart materials in various biomedical fields.

[0069] The present invention demonstrates the formation of a self-assembled vesicle structure (below the LCST of elastin and the UCST of regilin).

[0070] In the present invention, a biblock of a copolypeptide composed of six repeating units of resilin and elastin, respectively, was fused with the anti-angiogenic hexapeptide anti-Flt1 at the N-terminus using molecular cloning, and with high yield E.coliIt was overexpressed in and purified using the LCST of the elastin block by reverse transition cycle (ITC). The inventors found that anti-Flt1 functionalized elastin and regilin-based vesicles can competitively bind to vascular endothelial growth factor (VEGFR1) through non-covalent interactions in the presence of other growth factors such as vascular endothelial growth factor (VEGF) and can be used as potent inhibitors of angiogenesis.

[0071] In particular, the biblock that self-assembles into vesicles was selected because the surface area of ​​the vesicle is relatively larger than that of the micelle, allowing it to incorporate more functional epitopes and improve binding affinity. Additionally, it was shown that the hydrophobicity of anti-Flt reduces the transition temperature. Therefore, among the vesicle-forming biblocks, E6R6 was selected to overcome the fusion effect of anti-Flt and prevent total aggregation of the polypeptide at body temperature. The present invention demonstrates that regilin and elastin, which are biocompatible protein-based bilayer vesicle structures, can be potential candidates for therapeutic targeted delivery systems. Furthermore, the fusion of anti-Flt1 with elastin and regilin can overcome problems such as the high polydispersity index and inconsistent molecular weight distribution associated with the conjugation of anti-Flt1 with hyaluronic acid (HA).

[0072] Smart self-assembled bionanomaterials consisting of fusion block copolypeptides possessing multiple functionalities are being developed for various applications in fields related to improved drug delivery, sensing, and biomedical imaging. In particular, nano-vesicles (NVs) developed from stimulus-responsive genetically engineered amphiphilic polypeptides are becoming increasingly important materials because the vesicles' larger surface area supports multiple or higher valence, as well as increased biocompatibility and biodegradability. Peptide-based therapeutics promise highly specific and active targeting for individual receptors but have relatively short half-lives.

[0073] To address this problem, as described in the following examples, the inventors developed a therapeutic NV possessing angiogenic properties and excellent biocompatibility through the dynamic self-assembly of an elastin- and resilin-based block copolypeptide (EBP-RBP) functionalized with an anti-angiogenic peptide (anti-Flt1) genetically fused to the N-terminus. Anti-Flt1 (hexapeptide) is a novel antagonist of vascular endothelial growth factor receptor 1 (VEGF) and competitively binds to VEGFR1 in the presence of VEGF. AF1-E6R6 is E.coliIt was overexpressed and purified in high yield (~500 mg / L culture) by inverse transition cycling of the EBP block. It exhibits a convertible transition from vesicles to micelles due to alternating aggregation between RBP or EBP blocks as a function of temperature. Additionally, concentration, pH, ionic strength, and heating rate-dependent thermal profiles were performed to demonstrate the effects of anti-Flt1 fusion on the LCST and UCST behavior of EBP and RBP blocks. Compared to the E6R6 biblock, AF1-E6R6 showed a decrease in LCST and an increase in UCST due to changes in the ratio of hydrophobic to hydrophilic blocks, whereas both blocks followed similar trends with respect to pH, ionic strength, and heating rate. AF1-E6R6 demonstrated concentration and culture-dependent intracellular localization in HUVEC through receptor-ligand mediated interactions of anti-Flt1 and VEGFR1. The anti-angiogenic efficacy of anti-Flt1 mounted NVs was confirmed in the presence of VEGF by an analysis of HUVEC tubular formation, and AF1-E6R6 showed a dose-dependent reduction in tubular formation. In summary, AF1-E6R6-based NVs have the potential for specific targeting and downregulation of VEGFR1 for the treatment of various diseases such as diabetic retinopathy.

[0075] The therapeutic composition comprising the fusion polypeptide for inhibiting angiogenesis according to the present invention is a pharmaceutical composition. The pharmaceutical composition may include the fusion polypeptide and other substances that do not interfere with its use for inhibiting angiogenesis in vivo. These other substances are not limited and may include diluents, excipients, carriers, and / or other angiogenesis-inhibiting substances.

[0076] In some embodiments, the fusion polypeptide for inhibiting neovascularization of the present invention is formulated for ordinary human administration by being formulated with a suitable diluent, for example, comprising sterile water and ordinary saline solution.

[0077] The administration or delivery of the therapeutic composition according to the present invention may be via any route available to the target tissue through that route. For example, administration may be by direct injection into the target tissue (e.g., heart tissue), such as local or intradermal, subcutaneous, intramuscular, intraperitoneal, intra-arterial, intra-coronary, intradural, or intravenous injection, or intravitreal injection. The stability and / or efficacy of the fusion polypeptide disclosed in the present invention take into account convenient routes of administration including subcutaneous, intradermal, intravenous, and intramuscular.

[0078] The present invention provides a method for delivering a fusion polypeptide to cells (e.g., as part of a composition or formulation described in the present invention) and a method for treating, alleviating, or preventing the progression of a disease in a subject. As used in the present invention, the terms “subject” or “patient” mean any vertebrate, including but not limited to humans and other primates (e.g., chimpanzees and other ape and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild, and competitive birds such as chickens, turkeys, and poultry, ducks, geese, etc.). In some embodiments, the subject is a mammal.

[0079] In another embodiment, mammals are humans.

[0080] The fusion polypeptide or pharmaceutical composition of the present invention is a target cell (e.g., mammalian cell) and in vitro or in vivo It can be contacted.

[0081] In another aspect, the present invention provides a method for treating or preventing a disease caused by neovascularization, comprising the step of administering a therapeutic composition according to the invention to an individual.

[0082] For clinical use, the fusion polypeptide of the present invention may be administered alone or formulated into a pharmaceutical composition via any suitable route of administration effective in achieving the desired therapeutic result. The "route of administration" of the oligonucleotide of the present invention will mean enteral, parenteral, and topical administration or inhalation. Enteral routes of administration of the fusion polypeptide of the present invention include the oral cavity, gastrointestinal tract, and rectum. Parenteral routes include ocular injection, intravenous, intraperitoneal, intramuscular, intrathecal, subcutaneous, topical injection, vaginal, topical, nasal, mucosal, and pulmonary administration. Topical routes of administration of the fusion polypeptide of the present invention refer to external application of the oligonucleotide into the epidermis, oral cavity and ears, eyes, and nose.

[0083] The above therapeutic composition may be administered by parenteral, oral, transdermal, sustained-release, controlled-release, delayed-release, suppository, catheter, or sublingual administration.

[0084] The fusion polypeptide in the above therapeutic composition may be administered in an amount of 15 mg / kg or less by intravenous injection and 2.5 mg or less by intravitreal injection when administered in combination with other drugs.

[0086] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0088] [Example]

[0089] Example 1. Materials

[0090] pET-21a(+) vector and BL21(DE3) E. coli cells Purchased from Novagen Inc. (Madison, WI, US). TOP10 Competent Cells and Calcein-AM were purchased from Invitrogen (Carlsbad, CA, US). HUVECs were purchased from the American Type Culture Collection (ATCC) (Virginia, US). All custom oligonucleotides were synthesized at Cosmo Gene Tech (Seoul, South Korea). Recombinant human VEGF-165 (rhVEGF 165...was purchased from Sino Biological Inc. (Beijing, China). Restriction endonucleases, including FastAP thermosensitive alkaline phosphatase, BamHI, and XbaI, were purchased from Fermentas (Ontario, Canada). BseRI, AcuI, and T4 DNA ligase obtained from New England Biolabs (Ipswich, MA, US) were purchased from Elpis Bio-tech (Tajeon, South Korea). DNA miniprep, gel extraction, and PCR purification kits were purchased from Geneall Biotechnology (Seoul, South Korea). Dyne Agarose High was purchased from DYNE BIO, Inc. (Seongnam, South Korea). All Top10 cell cultures were cultured in TB DRY medium purchased from MO BIO Laboratories, Inc. (Carlsbad, CA, US). All BL21(DE3) cell cultures were grown in Circle Grow medium obtained from MP Biomedicals (Solon, OH, USA). The precast gel Ready Gel (Tris-HCl 2-20%) was purchased from Bio-Rad (Hercules, CA, USA). Phosphate-buffered saline (PBS, pH 7.4), ampicillin, polyethyleneamine (PEI), bovine serum albumin (BSA), and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Matrigel was purchased from BD Biosciences (San Diego, CA, USA).

[0092] Example 2. Gene composition of AF1-E6 R6 block copolypeptide

[0093] A pair of oligonucleotides encoding the anti-Flt1 peptide (SEQ No. 1), which acts as a VEGFR1 antagonist, was chemically synthesized at Cosmo Genetech (Seoul, South Korea) and annealed into an oligonucleotide cassette having cohesive ends of AcuI and BseRI. The oligonucleotide cassette encoding the anti-Flt1 peptide was rationally designed without recognition sites for BseRI, XbaI, AcuI, and BamHI for seamless gene cloning. Genes for the fusion polypeptide of AF1-E6R6 block diblock copolypeptides were generated using plasmids containing the E6R6 and anti-Flt1 oligonucleotide cassettes having recognition sites for BseRI, XbaI, AcuI, and BamHI (see Table 1 for sequence information). First, the oligonucleotide pair encoding anti-Flt1 was heated at 95°C for 2 minutes with 50 μL of T4 DNA ligase buffer at a concentration of 2 μM oligonucleotides, and then annealed by slowly cooling the reaction mixture to room temperature over 3 hours. To molecularly clone AF1-E6R6, the plasmid vector encoding E6R6 was digested with 15 U of BseRI in CutSmart buffer at 37°C for 30 minutes. The restriction plasmid was purified using a PCR purification kit and then dephosphorylated with 10 U of FastAP, a thermosensitive alkaline phosphatase, in CutSmart buffer at 37°C for 1 hour. The restricted and dephosphorylated plasmid DNA was purified using a PCR kit and eluted in 40 μL of distilled and deionized water. Ligation was performed by incubating 90 pmol of purified insert with 30 pmol of linearized vector and 1 U of T4 DNA ligase buffer at 16°C for 30 minutes. The product was then transformed into TOP10 competent cells and plated on SOC plates supplemented with 50 μg / mL of ampicillin.Transformers were initially screened by diagnostic restriction digestion on an agarose gel and further confirmed by DNA sequencing.

[0095] Example 3. Anti-Flt1-EBP[G 1 A 3 F 2 ] 6 -RBP[m-Dros] 6 Expression and Purification of Diblock Copolypeptides

[0096] E6R6 and AF1-E6R6 gene-containing plasmids E.coli BL21 (DE3) cells were transformed. A single bacterial colony was inoculated into 10 mL of CG medium (primary culture) containing 50 μg / mL ampicillin and grown overnight at 180 rpm. The cells were cultured in [location unclear]. 400 mL of CG medium supplemented with 50 μg / mL ampicillin was inoculated as the primary culture into a 2 L flask and incubated at 200 rpm for 6 hours at 37°C. 15 mL of the secondary culture was inoculated into 500 mL of CG (50 μg / mL ampicillin) in a 2 L flask, and after 3 hours of incubation, IPTG was added to achieve a final concentration of 1 mM to induce expression. Bacterial cells were harvested after 12 hours by centrifugation, and the cell pellet was resuspended in PBS with 1.5 M urea. Cell lysis was obtained by sonicating the sample (VC-505, Sonic and materials Inc, Danbury, CT) in an ice bath for 5 minutes at 60% power (10 seconds on, 30 seconds off). E6R6 and AF1-E6R6 diblock copolypeptides were purified by inverse transition cycle (ITC). During ITC, cellular debris was separated by centrifugation at 16,000 rpm for 30 minutes at 4°C, and soluble lysates were transferred to a new tube. Nucleic acid contaminants were separated by centrifugation at 16,000 rpm for 15 minutes at 4°C. NaCl was added to the samples to a final concentration of 3 M to induce phase transition of the fusion proteins. Aggregated fusion proteins were separated by centrifugation at 16,000 rpm for 20 minutes at 40°C. The aggregated fusion proteins were resuspended in cold PBS containing 1.5 M urea, and the samples were centrifuged at 16,000 rpm for 15 minutes at 4°C to remove remaining insoluble material. This aggregation and resuspension process was repeated 5 to 7 times until the purity of the fusion proteins was obtained. The purified sample was dialyzed against deionized water to remove urea and salts, and freeze-dried for further characterization.

[0098] Example 4. Analysis of the physicochemical properties of block copolypeptides

[0099] The purity and molecular weight of all polypeptides were analyzed by SDS-PAGE stained with copper chloride solution (0.3 M). The phase transition behavior of the polypeptides was analyzed as a function of temperature from 10°C to 90°C in PBS (10 mM, pH 7.4) at different heating and cooling rates of 1°C / min at 350 nm (OD) 350 Optical density was measured at ) and characterized using a UV-visible spectrophotometer. T t θ is defined as the temperature at which the first derivative of turbidity is maximum as a function of temperature. The effects of concentration and salt on the thermal behavior of polypeptides in PBS were investigated at a heating rate of 1 °C / min; concentrations varied from 12.5 to 100 μM, and NaCl concentrations varied from 0.125 M to 1.5 M NaCl (polypeptide concentration was 25 μM). The size of the polypeptides was measured via DLS at different temperatures with a scattering angle of 90°. The hydrodynamic radii (R) of the E6R6 and AF1-E6R6 blocks h ) was obtained in PBS at 37°C and a concentration of 25 μM. The sample was left in equilibrium for 10 minutes before light scattering occurred, and 11 measurements were performed.

[0101] Example 5. E 6 R 6 and AF1-E 6 R 6 Nanoscale image of block copolypeptide

[0102] Transmission electron microscopy (TEM) analysis was performed using a JEM-2100F FE-STEM (JEOL, Germany) operating at acceleration voltages of 80–200 kV to verify whether nanostructures were formed as a result of the self-assembly of polypeptides according to thermal reactivity. The TEM grid was immersed in 25 μM of sample, dried on filter paper, and then stained with 2% phosphotungstic acid. Samples were prepared at 37°C to confirm the self-assembled structure.

[0103] Self-assembled vesicles exist in a colloidal state. Therefore, to better understand the vesicles, images in the swollen state provide more accurate details regarding nanostructural dimensions. Considering the application of these vesicles at body temperature, CSLM samples were prepared at 37°C. E6R6 and In PBS, the AF1-E6R6 block It was dissolved at 0.8 mg / ml to make the final concentration 25 μM. Prior to confocal microscopy, the sample was equilibrated at 37°C. CLSM images were acquired in the swollen state using a He / Ne and argon laser (Leica TCS SP) in bright-field mode. A heating step was used to maintain the temperature at 37°C.

[0105] Example 6. E 6 R 6 and AF1-E 6 R 6 Internalization of base vesicles

[0106] For internalization analysis, E6R6 and AF1-E6R6 were conjugated with FITC (fluorescein isothiocyanate), purified, and dialyzed under basic conditions, and lyophilized for further experiments. 4.5X10 4HUVECs were cultured overnight on a Lab-Tek II Chamber Slide, and the medium was replaced with fresh medium containing a sample at a final concentration of 1 μM. After 4 hours of incubation, the medium was removed, and the cells were washed three times with medium (without phenol red) to remove unbound nanoparticles. Internalization of the vesicles was observed using a confocal laser scanning microscope (CLSM).

[0108] Example 7. HUVEC using anti-Flt1-EBP-RBP diblock copolypeptide In a test tube Tubing analysis

[0109] HUVEC test using E6R6 and AF1-E6R6 block copolypeptides Within the jurisdiction Tubing assays were performed to evaluate the effects of E6R6 and AF1-E6R6 block-based bilayer vesicles on endothelial cell proliferation, migration, and tubular formation. 150 μL of 8.7 mg / ml Matrigel for coating was coagulated by incubating in a 48-well plate at 37°C for 1 hour. HUVECs were cultured with 10 μM. For fluorescence labeling, HUVECs were cultured with 10 μM Calcein-AM at 37°C for 15 minutes and washed several times with PBS. 4 x 10 4 Calcein-labeled HUVECs were grown in Matrigel-coated wells and 50 ng / ml of recombinant human rhVEGF 165We measured how endothelial cell proliferation, migration, and tubular formation were stimulated by incubating with E6R6 and AF1-E6R6 block copolypeptides of specific antagonists at various concentrations for 4 hours at 37°C. The E6R6 block was used as a control to clearly demonstrate how much the AF1-E6R6 block copolypeptide structure could inhibit tubular formation in HUVECs. Tubular formation in HUVECs was quantified based on the migration of HUVECs after 4 hours of incubation in PBS, by taking images with a Micromanipulator (Olympus, Tokyo, Japan) and measuring the total tube length in three random fields per well using Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).

[0111] Example 8. E 6 R 6 and AF1-E 6 R 6 Cytotoxicity of base vesicles

[0112] For cytotoxicity analysis, 12 x 10 4 HUVECs at a concentration of 100 µl / cell / well were cultured overnight in EBM-2 96-well plates to allow the cells to attach to the bottom of the wells. After 12–16 hours, the medium was removed, fresh medium containing the samples was added, and the plates were incubated at 37°C for 4 hours. After adding EBM-2 medium containing 10% WST solution, absorbance was measured at 495 nm after 2–4 hours and calculations were performed.

[0113] Anti-Flt1 fusion elastin and resilin block copolypeptides were synthesized using recombinant DNA technology, where the notations for elastin and resilin block are (E), respectively. n and (R) n..., where "n" indicates the number of repeating units. Alanine, glycine, and phenylalanine in a 1:3:2 ratio consist of 6 pentapeptides (Val-Pro-Ala-X aa - Gly) was used as a guest residue (Xaa) in a hydrophobic elastin block containing repeat units. A hydrophilic resilin block nearly identical to naturally occurring resilin consists of two resilin repeat sequences, GGRPSDSYGAPGGGN and GGRPSSSYGAPGQGN (total 30 amino acids), and was designed to maintain the UCST behavior of resilin and the LCST behavior of elastin by considering the sensitivity of the resilin block to the sequence and the number of repeat units of recombinant resilin. The (E)6 and (R)6 blocks were each fused with six repeat units and self-assembled into bilayered hollow vesicles. A small functional hexapeptide with anti-angiogenic properties, namely anti-Flt1, was genetically fused with the E6R6 block instead of conjugation to overcome disadvantages such as inconsistent molecular weight distribution. Figure 1(A) schematically illustrates the molecular designs of the anti-Flt1 (AF1) amino acid sequence, the elastin-based block (E), and the regilin-based block (R). To clearly observe the effects of anti-Flt1 on angiogenesis, an elastin and regilin-based biblock without anti-Flt1 was selected as a control. For the E6R6 block (control), a single block of elastin and regilin was synthesized using recursive directional ligation of up to six repeat units. A biblock with elastin at the N-terminus and regilin at the C-terminus was synthesized by ligation of the single blocks. For the synthesis of AF1-E6R6, AF1 was fused to the N-terminus of E6R6. . E6R6 and The self-assembly of AF1-E6R6 into a bilayer is shown in Fig. 1 (B). The resilin block used in the present invention exhibits aggregation behavior due to non-covalent interactions below the upper critical solution temperature (UCST), whereas the elastin block exhibits solubility behavior below the lower critical solution temperature (LCST). E6R6 and The AF1 - E6R6 block self-assembles into vesicles due to the aggregation of the resilin block below the LCST and UCST. Specifically, the resilin aggregates below the UCST, and the EBP self-assembles into vesicles while remaining soluble below the LCST. FIG. 1(A) is The swelling state (CLSM) image of AF1-E6R6 on PBS clearly shows the self-assembly of vesicles. Fusion of anti-Flt1 at the N-terminus of the E6R6 block did not affect the vesicle-like self-assembly of this block. Figure 1(B) is a schematic diagram of AF1-E6R6-based vesicles for in vitro targeting of vascular endothelial growth factor receptor 1 (VEGFR1).

[0114] Figure 1(C) shows the results of observing the effect of vesicles on angiogenesis in human umbilical vein endothelial cells (HUVEC) by performing an in vitro tubing analysis on AF1-E6R6. . As shown in Fig. 1(A), anti-Flt1 functionalized vesicles were formed at physiological temperatures (body temperature range), exhibiting anti-angiogenic properties indicated by the limited migration and tubular formation ability of HUVECs in the anti-Flt1 functionalized treated samples. Figure 1(D) shows the intracellular location of AF1-E6R6 vesicles compared to E6R6, which does not show binding to the cell.

[0115] The AF1-E6R6 fusion polypeptide was constructed using molecular cloning via the fusion of the anti-Flt1 gene and the E6R6 block gene, and was synthesized and multimerized by known recursive directional ligation (RDL). For the synthesis of AF1-E6R6, a pair of anti-Flt1 oligonucleotides without recognition sites for seamless cloning were designed and chemically synthesized at Cosmo Genetech (Seoul, Korea). In the first step, the anti-Flt1 oligonucleotide was annealed by heating 50 μl of oligonucleotides at a concentration of 2 μM contained in T4 DNA ligase at 95 °C for 2 minutes, followed by slowly cooling the solution to room temperature over 3 hours. The pET21a (+) vector containing the E6R6 gene was cleaved with BseRI, and the anti-Flt1 nucleotide cassette was inserted. E6R6 and (control group) after BamHI and XbaI restriction and The DNA agarose gel of AF1-E6R6 is shown in Figure 2 (A-i). The DNA size of the standard marker and the estimated DNA size of the E6R6 and AF1-E6R6 genes are shown on the left and right, respectively. Both the E6R6 and AF1-E6R6 blocks were overexpressed in circular growth medium with IPTG induction for 15 hours. Inverse transition cycling (ITC) was used to purify both blocks by utilizing the LCST behavior of the E6 block. Aggregation occurred due to thermally induced transition of the E6 block above the LCST, and it was resolubilized in PBS at a temperature lower than the LCST. During the resuspension of the AF1-E6R6 aggregates, 1.5 M urea was added to PBS to completely dissolve the AF1-E6R6 block, as the R6 block exhibits UCST behavior below the LCST of E6. Above the LCST, the E6 block formed aggregates, thus hindering the complete dissolution of AF1-E6R6. After 5–7 rounds of ITC, the E6R6 block was purified in a yield of ~20 mg per liter of culture. On the other hand, the AF1-E6R6 block was purified in a yield of over 500 mg per liter of culture; this is because the difference in the N-terminal sequence of the AF1-E6R6 block allows for the regulation of expression levels depending on the fusion sequence of the elastin blocks. Copper-stained SDS-PAGE gel images of the E6R6 and AF1-E6R6 blocks are shown in Figure 2(A-ii). The purity of the polypeptides was confirmed by size exclusion chromatography. Both the E6R6 and AF1-E6R6 blocks were completely dissolved in 1.5 M urea in PBS at a concentration of 5 mg / ml. 1.5 M urea was sufficient to overcome resilin-associated aggregation. The absorbance of the polypeptides was measured at 280 nm. Both E6R6 and AF1-E6R6 As shown in Fig. 2A (iii), the presence of a sharp single peak indicates that it is highly purified. To observe the thermal transition behavior of the E6R6 and AF1-E6R6 blocks, both blocks were dissolved in 10 mM PBS at concentrations of 25 μM, 50 μM, and 100 μM, and thermal characterization was performed by measuring absorbance at 350 nm at a heating rate of 1 °C per minute. The LCSTs of the E6R6 and AF1-E6R6 blocks were recorded as 55.7 °C and 40.0 °C, respectively, for the 25 μM sample, as shown in Fig. 2(B). At a polypeptide concentration of 25 μM, the UCST of the E6R6 block was recorded at 88.5 °C, while the UCST of the AF1-E6R6 block was higher than 90.0 °C. The hydrophobicity of anti-FLT1 increased the overall hydrophobic-to-hydrophilic ratio of the polypeptide, which consequently shifted the UCST to a much higher value. Figure 2(C) showed that when a heating rate of 0.5 C / min was used, the UCST of the AF1-E6R6 block increased to 86.5°C compared to the E6R6 block, confirming the sensitivity of UCST to the heating rate. By introducing the hydrophobic AF1 to the N-terminal group, the LCST decreased while the UCST increased compared to the E6R6 block. The UCST behavior of AF1-E6R6, which was not observed at a heating rate of 1°C / min, was observed even at lower heating rates. The fusion of anti-FLT1 resulted in a decrease in LCST and an increase in UCST. The decrease in LCST is due to the hydrophobicity of the N-terminal anti-FLT1 sequence. The decrease in LCST may be due to increased hydrophobic interactions provided by anti-Flt1. An increase in the proportion of hydrophobic blocks can increase LCST by stabilizing resilin aggregation even at high temperatures. The concentration-dependent profile in Figure 2(B) shows that LCST decreased in both the E6R6 and AF1-E6R6 blocks as the concentration increased.UCST is sensitive to the hydrophilic-to-hydrophobic ratio and length, whereas it is independent of concentration and is represented by the unchanged UCST for both the E6R6 and AF1-E6R6 blocks. To observe the effect of pH on UCST, the effect of pH was measured. Polypeptides were prepared at a concentration of 25 µM in phosphate buffers at pH 6.4 (weakly acidic), 7.4 (physiologically relevant), and 8.4 (weakly basic). Figure 2(D) shows the pH-dependent thermal profiles of both the E6R6 and AF1-E6R6 blocks. UCST decreased in both blocks at the baseline pH (8.4), because the pH arginine residues were deprotonated, altering the solubility of the resilin(R) block. Compared to UCST, LCST exhibits higher sensitivity to chaotropic salts. To confirm the salt-dependent effects on E6R6 and AF1-E6R6 blocks, salt-dependent thermal profiles were measured using a constant polypeptide concentration and various NaCl concentrations ranging from 0.01 to 0.1 M. Figure 2(E) indicates that the LCST is not affected by various salt concentrations. The LCSTs of both polypeptides were slightly lowered, which means that slight changes in salt concentration do not have a significant effect on the LCST.

[0116] Additionally, transmission electron microscopy (TEM) was performed to confirm the self-assembly and morphology of the two blocks of spherical vesicles in a dry state. TEM samples were prepared at an equal concentration of 25 μM. First, the TEM samples were equilibrated in PBS at 37°C, and then the prepared samples were dried at 37°C prior to measurement. The TEM samples were prepared on a carbon-coated grid (equilibrated at 37°C), and a 2% (w / v) PTA staining solution (equilibrated at 37°C) was used to produce better contrast and visualization on the carbon-coated grid. Figures 3 (A-i, ii) and 3 (B-i, ii) show TEM images of the E6R6 and AF1-E6R6 blocks, respectively. Figure 3 contains transmission electron microscopy (TEM) images to confirm the morphology of the nanostructures and a statistical analysis of the sizes of the E6R6 (control) and AF1-E6R6 block vesicles. (A-i), (A-ii) and (B-i), (B-ii) are TEM images of the E6R6 and AF1-E6R6 blocks, respectively. TEM samples were prepared at 37°C, and 2% (w / v) tungstic acid was used as a negative stain. (C) shows the statistical analysis of the vesicle size distribution in TEM for E6R6 and AF1-E6R6. Image J was used to measure the average vesicle size for the two blocks by randomly selecting particles from the TEM and SEM images; the average diameter of E6R6-based vesicles in TEM was 281.8 nm, while the average diameter of AF1-E6R6-based vesicles was 233.3 nm. The fusion of anti-Flt1 and the E6R6 block resulted in slightly smaller vesicles. This reduction in AF1-E6R6-based vesicle size is due to an increase in the ratio of hydrophobic to hydrophilic blocks. The reduction in nanostructure size in both E6R6 and AF1-E6R6-based vesicles compared to the hydrodynamic radius is attributed to the dry state preparation of the vesicles.

[0117] To confirm the self-assembly of E6R6 and AF1-E6R6 blocks into nanostructures, dynamic light scattering (DLS) experiments in soluble states were performed on both samples (25 μM) at 37°C. Hydrodynamic radius (R h ) was measured at 37°C in PBS at a concentration of 25 µM at a scattering angle of 90 degrees. R of E6R6 and AF1-E6R6 blocks h As shown in Fig. 4(A), the dimensions were 533.2 nm (Std. dev 118.7) and 365.3 nm (Std dev 97.01), respectively. The size reduction of the AF1-E6R6 block was due to a slight increase in the proportion of hydrophilic blocks below the LCST and UCST; as the proportion of hydrophilic blocks increases, the nanostructure becomes smaller and dimensionality is formed. The addition of smaller peptides to the N-terminus of the hydrophilic blocks increased the volume of solvent directed toward the blocks, resulting in an overall reduction in vesicle size. Temperature-dependent measurements of the hydrodynamic radii of the two blocks demonstrated the effect of temperature on the Z-mean of the nanostructures of the two blocks (data not shown). The E6R6 block is It exhibits a z-mean range of approximately 1 μm in diameter below the LCST. In contrast, the AF1-E6R6 block maintains overall solubility at low temperatures and maintains solubility in nanostructures as the temperature increases. This is because while the overall increase in hydrophobicity of the block copolypeptide dominates the nanostructure-forming ability of the R6 block, the overall kinetics favor nanostructure formation below the LCST as the temperature increases. Both blocks show total aggregation with a rapid increase in size above the LCST. The influence of temperature on the count rate of nanostructures formed by both blocks was also observed (data not shown). The trend in count rates is similar to the Z-mean, as the average count rate of the E6R6 block is approximately 120 kcps lower than the LCST, while the average count rate of the AF1-E6R6 block increases with increasing temperature below the LCST. Above the LCST, both blocks exhibit total aggregation, indicated by a rapid increase in counts. The fusion effect of AF1 on the E6R6 block was further utilized by first verifying the colloidal state at physiological temperature. To better understand the bilayer vesicle structure of the swollen E6R6 and AF1-E6R6 blocks, both samples were resuspended in PBS at a final concentration of 25 μM. Prior to confocal measurement, both samples were equilibrated at 37°C to ensure self-assembly for both blocks, and imaging was performed at a 37°C heating stage to confirm self-assembly of vesicles at body temperature as shown in Fig. 4(C). The AF1-E6R6 block forms vesicles, and the size of these vesicles is slightly reduced compared to the vesicles of E6R6. This clearly confirms that the length ratio of the hydrophobic and hydrophilic blocks is a key factor in determining the final nanostructure size. Fig. 4(E) is This shows the statistical analysis of E6R6 and AF1-E6R6-based vesicles in a swollen state. Image J was used to measure the average size of vesicles for the two blocks by randomly selecting particles from confocal laser scanning microscopy (CLSM) images, and the average sizes of the E6R6 and AF1-E6R6 blocks were calculated to be 2.84 μm and 2.01 μm, respectively. Compared to vesicles based on the swollen AF1-E6R6 block, the E6R6 block-based vesicles are larger, demonstrating that anti-Flt1 fusion and increased overall hydrophobicity influence the vesicle size. Figure 4(D) shows FITC-labeled vesicles of the AF1-E6R6 block. The vesicles clearly show an outer layer and a hollow core.

[0118] Calcein-free HUVECs were treated with samples of E6R6 and AF1-E6R6 conjugated with FITC (fluorescein isothiocynate) to locate the intracellular positions of these vesicles (Fig. 5(A)). For the conjugation of E6R6 and AF1-E6R6, lyophilized samples were resuspended in sodium bicarbonate buffer, and the conjugation reaction was performed at low temperature under dark conditions, followed by purification via multiple cycles of ITC. Completely purified and dialyzed samples were lyophilized for further analysis. FITC-bound samples were resuspended in PBS at a concentration of 1 μM for 4 hours, and confocal laser scanning imaging was performed to monitor the positions of the vesicles. After 4 hours AF1-E6R6 clearly demonstrated internalization resulting from the interaction between anti-Flt1 and VEGFR1, whereas E6R6 did not exhibit binding or internalization (Figs. 5(B) and 5(C)). VEGFR can follow various internalization pathways, the most common of which is clathrin-mediated endocytosis. However, in the presence of VEGF, micropinocytosis is a more prominent pathway for VEGFR2 internalization and is known to play an important role in endothelial cell function. Therefore, VEGFR1 exhibits internalization in the presence of anti-Flt1. Furthermore, since interactions can increase with increasing valence, multiple valence plays an important role in receptor-ligand interactions. AF1-E6R6-based vesicles, possessing a larger surface area and enhanced multivalentity, can increase the interaction between anti-Flt1 and VEGFR1, leading to internalization. Another reason why cell absorption of large vesicle structures is possible is that the introduction of arginine-rich peptides enhances cell absorption of polypeptides, which occurs due to the presence of arginine-rich resilin blocks.

[0119] HUVEC's E6R6 to evaluate the potential toxicity of polypeptides 및Cytotoxicity analyses were performed on both AF1-E6R6 block copolypeptides. Figure 6(A) clearly shows that the viability of HUVECs after 2–4 hours of incubation is unaffected by E6R6 and AF1-E6R6. Figure 6(B) clearly shows that the viability of HUVECs after 2–4 hours of incubation is unaffected by FITC-conjugated E6R6 and AF1-E6R6. After 4 hours of incubation, the E6R6 block exhibited the highest viability at a concentration of 0.1 μM in both FITC-conjugated and non-conjugated samples, whereas the AF1-E6R6 block showed the highest viability at a concentration of 0.25 μM in both FITC-conjugated and non-conjugated samples. The high cell viability of AF1-E6R6-based vesicles demonstrates their potential as potential therapeutic candidates for anti-angiogenic applications. .

[0120] To observe the in vitro anti-angiogenic activity of AF1-E6R6-based vesicles, tubing assays of E6R6 and AF1-E6R6 blocks were performed on HUVEC motility at optimized concentration ranges while maintaining cell viability. Calcein-labeled HUVECs (4x10⁶) were used in Matrigel-coated 48-well plates. 4 Cells / well) were grown. Two control groups were used. 1) The first control group was 50 ng / ml rhVEGF 165 1) containing HUVEC together and 2) the second control group is 50 ng / ml rhVEGF 165and includes HUVECs with E6R6-based vesicles. Figure 7(A) shows fluorescence images of calcein-labeled HUVECs of both E6R6 and AF1-E6R6 blocks. Compared to the tubular formation ability (0%) in HUVECs cultured without VEGF, HUVECs cultured with 50 ng / ml VEGF showed high tubular formation ability (100%) induced by VEGF signaling. A clear difference in tubular formation ability can be observed in cells containing AF1-E6R6-based vesicles. The migration and tubular formation abilities of AF1-E6R6-treated HUVECs were significantly reduced compared to the control group. Figure 7(B) shows 50 ng / ml rhVEGF 165 This shows E6R6 and AF1-E6R6 at various concentrations treated with HUVECs. Fluorescence images and tube lengths of HUVECs were used to calculate the effects of E6R6 and AF1-E6R6 using ImageJ software. AF1-E6R6 at various concentrations demonstrates high tube formation inhibition ability compared to E6R6-treated HUVECs in the same concentration range, more specifically in the 0.25 μM–1 μM range. The highest tube formation inhibition ability is observed in AF1-E6R6-treated vesicles at a concentration of 0.75 μM.

[0121] The anti-Flt1 fusion resilin elastin-based block copolypeptide demonstrates a very high ability to reduce tubular formation as anti-Flt1 competitively binds to VEGFR1. Therefore, AF1-E6R6 can be used as a potential therapeutic candidate for anti-angiogenesis associated with various types of cancer.

[0123] The description of the invention set forth above is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A self-assembled nanovesicle structure based on a fusion polypeptide for inhibiting angiogenesis, comprising an anti-FLT1 peptide represented by SEQ ID NO. 1; an elastin-based polypeptide (EBP) represented by SEQ ID NO. 2 linked to the peptide; and a resilin-based polypeptide (RBP) represented by SEQ ID NO. 3 linked to the EBP, wherein the structure is formed by self-assembling into a double-layer nanovesicle of an anti-FLT1 layer and an EBP-RBP layer at a temperature below the lower threshold solution temperature (LCST) of the EBP and below the upper threshold solution temperature (UCST) of the RBP. Claim 2 A polypeptide-based self-assembled nanovesicle structure for inhibiting angiogenesis according to claim 1, wherein at a temperature below the LCST of the EBP and below the UCST of the RBP, the anti-FLT1 forms a surface layer, the RBP aggregates, and the EBP becomes soluble, thereby self-assembling into a double-layer nanovesicle of the anti-FLT1 layer and the EBP-RBP layer. Claim 3 A polypeptide-based self-assembled nanovesicle structure for inhibiting neovascularization according to claim 1, wherein the temperature below the LCST of the EBP and below the UCST of the RBP is within the body temperature range. Claim 4 A polypeptide-based nanovesicle structure for inhibiting angiogenesis, characterized in that, in claim 1, the nanovesicle structure has a multivalent anti-FLT1 peptide. Claim 5 A composition for treating a disease caused by neovascularization comprising a self-assembled nanovesicle structure according to any one of claims 1 to 4, wherein the disease caused by neovascularization is one or more selected from diabetic retinopathy, retinopathy of prematurity, macular degeneration, choroidal neovascularization, neovascular glaucoma, ocular disease caused by corneal neovascularization, rejection reaction during corneal transplantation, corneal edema, corneal opacity, cancer, hemangioma, angiofibroma, rheumatoid arthritis, and psoriasis. Claim 6 delete Claim 7 A theranostic nanoprobe for a disease caused by neovascularization, comprising: a fluorescent dye; and a self-assembled nanovesicle structure of any one of claims 1 to 4; wherein the disease caused by neovascularization is one or more selected from diabetic retinopathy, retinopathy of prematurity, macular degeneration, choroidal neovascularization, neovascular glaucoma, ocular disease caused by corneal neovascularization, rejection reaction during corneal transplantation, corneal edema, corneal opacity, cancer, hemangioma, angiofibroma, rheumatoid arthritis, and psoriasis. Claim 8 delete

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