Fusion peptide comprising thrombus-targeting peptide, ferritin fragment and thrombolytic peptide, and use thereof

Abstract

The present invention relates to: a fusion peptide comprising a thrombus-targeting peptide, ferritin fragment and a thrombolytic peptide; and a use thereof and, more specifically, to: a fusion peptide in which a thrombus-targeting peptide, ferritin fragment and a thrombolytic peptide are sequentially linked; a composition for preventing or treating thrombotic disorders, containing the same as an active ingredient; a method for treating thrombotic disorders; and a therapeutic use. According to the present invention, CLT-sFt-μPn DCNC as a novel plasmin-based thrombolytic nanocage has: an effect of targeting a site at which thrombus is present; a low sensitivity to inhibitors present in the circulatory system; pharmacological activity strongly destroying both arterial and venous thrombi; and no side effects of bleeding, and thus can be very useful in developing an agent for preventing or treating thrombotic disorders.

Claims

1. A fusion peptide, comprising: (a) a clot-targeting peptide defined by the amino acid sequence of SEQ ID NO: 1; (b) a peptide defined by the amino acid sequence of SEQ ID NO: 3; and (c) any one peptide selected from the group consisting of microplasminogen defined by the amino acid sequence of SEQ ID NO: 5, and microplasmin defined by the amino acid sequence of SEQ ID NO: 6, wherein the peptides (a), (b), and (c) are sequentially linked.

2. The fusion peptide of claim 1, wherein the clot-targeting peptide (a) is linked to the N-terminus of the peptide (b) and the peptide (c) is linked to the C-terminus of the peptide (b).

3. The fusion peptide of claim 1, wherein the clot-targeting peptide (a) or the peptide (c) is linked to the peptide (b) via a linker.

4. A fusion peptide comprising the amino acid sequence of SEQ ID NO: 7.

5. A cage protein consisting of the fusion peptide of claim 1.

6. A polynucleotide encoding the fusion polypeptide of claim 1.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell transformed with the expression vector of claim 7.

9. A pharmaceutical composition for preventing or treating a thrombotic disease, the composition comprising the fusion peptide of claim 1 as an active ingredient.

10. The composition of claim 9, wherein the thrombotic disease is selected from the group consisting of acute myocardial infarction, ischemic stroke, hemorrhagic stroke, deep vein thrombosis, lower limb edema, acute peripheral arterial occlusion, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous sinus thrombosis, angina pectoris, cerebral infarction, and central retinal vein occlusion.

11. A method for treating a thrombotic disease in a subject in need thereof, the method comprising administering the fusion peptide of claim 1 to the subject in an amount effective for treating the thrombotic disease.

12. The method of claim 11, wherein the thrombotic disease is selected from the group consisting of acute myocardial infarction, ischemic stroke, hemorrhagic stroke, deep vein thrombosis, lower limb edema, acute peripheral arterial occlusion, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous sinus thrombosis, angina pectoris, cerebral infarction, and central retinal vein occlusion.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) Abbreviations used in all drawings are as follows:

(3) CLT: clot-targeting peptide, sFt: ferritin fragment, μPg: microplasminogen, μPn: microplasmin, CLT-sFt-μ: fusion protein in which respective peptides are linked.

(4) FIG. 1A is a diagram showing a schematic view of a fusion peptide monomer (CLT-sFt-μPg) according to the present invention and a cage formed by the monomers. The 3D structure of CLT-sFt-μPg was modeled using MODELLER v9.12 on the basis of the structures of human ferritin (PDB 2FG4) and microplasminogen (PDB 1QRZ). The CTL peptide (CNAGESSKNC (SEQ ID NO: 1)) is shown in red ribbon, the microplasminogen in green, and the ferritin fragment in light brown. The cleavage site (Arg.sup.561-Val.sup.562) by the plasminogen activator is shown in magenta, and the catalytic triad amino acid residues (His.sup.603, Asp.sup.646, and Ser.sup.741) are shown in orange color.

(5) FIG. 1B is a design of an assembly of CLT-sFt-μPg monomers. It can be confirmed that six 4-fold assemblies of CLT-sFt-μPg monomers are assembled to form a cage, and microplasminogen fused to the C-terminus of the ferritin fragment and CLT protein fused to the N-terminus thereof are exposed to the outside. The diameter of the cage protein consisting of CLT-sFt-μPg fusion peptides was calculated to be about 17 nm.

(6) FIG. 2A shows schematic diagrams of CLT-sFt-μPg and sFt-μPg, and shows SDS-PAGE results of the purified cages.

(7) FIG. 2B shows DLS analysis results of CLT-sFt-μPg DCNC and sFt-μPg DCNC.

(8) FIG. 2c shows transmission electron micrographic (TEM) analysis results of CLT-sFt-μPg DCNC and sFt-μPg DCNC.

(9) FIG. 3A shows schematic diagrams of the proteins used in the present invention. The flexible linker (GGGSG) is inserted between ferritin fragment and microplasminogen.

(10) FIG. 3B shows the results, wherein after the formation of clots, the FITC-labeled proteins were added on the clots at a concentration of 1.25 μM to monitor through a fluorescence microscope whether the proteins were bound to the clots. The clots were visualized under an optical microscope.

(11) FIG. 3C shows the results, wherein the FITC-labeled proteins (1 μM) were added to clots and then the amounts of bound proteins were quantified using a fluorescence spectrometer (ANOVA test, ****: P<0.0001, ***: P=0.0003).

(12) FIG. 3D shows the results, wherein CLT-sFt-μPg and sFt-μPg (1-4 μM) were added on clots and then the amounts of proteins bound to the clots were quantified.

(13) FIG. 4A shows the results of evaluating the degree of clot dissolution through turbidity, wherein the clots in 96-well plates were incubated with each of the proteins (7 μM) during the indicated times and then the absorbance was measured at 405 nm.

(14) FIG. 4B shows the results, wherein CLT-sFt-μPn or μPn was intravenously administered into mice and then the amount of α2-anti-plasmin in blood was measured.

(15) FIG. 4C shows the results, wherein CLT-sFt-μPn or μPn was intravenously administered into mice and then the time to stop bleeding on the wound of mouse tail ends was measured.

(16) FIG. 5A shows the results, wherein when respective proteins were treated following the blood occlusion by the induction of thrombosis in right central carotid artery of mice, the 12 transverse sections of the blood vessel were observed through H/E staining to investigate the degree of clot dissolution (RCCA: right central carotid artery).

(17) FIG. 5B shows the results, wherein the degree of occlusion of the blood vessel by clots was measured by the inForm program (PerkinElmer) and the area occupied by clots in each section relative to the overall vessel section area was quantified and normalized.

(18) FIG. 5C shows a diagram of a deep vein thrombosis animal model.

(19) FIG. 5D shows the results of naked-eye observation of clots remaining after treatment with each protein (top) and the results of measurement of each clot weight (bottom).

MODE FOR CARRYING OUT THE INVENTION

(20) Hereinafter, the present invention will be described in detail.

(21) However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

(22) <Methods>

(23) 1. Construction of Clot-Targeting Peptide (CLT)/Microplasminogen-Conjugated Double Chambered Nanocages and CLT-Microplasminogen Fusion Proteins

(24) The recombinant plasmids for expressing the double chambered nanocage (DCNC) and control protein were constructed using modified pET28 vector (Novagen). The modified pET28 contains extra cleavage sites of KpnI and NheI between NcoI/NdeI and an extra cleavage site of SpeI between Sa1I/XhoI. The gene encoding the short ferritin light chain (sFt) was prepared through PCT from cDNA of the human ferritin light chain, and as previously reported (ACS nano 7, 7462-7471, 2013), was incorporated between Nde1 and BamH1 for the expression in E. coli. The CLT protein (CNAGESSKNC)-encoding oligonucleotide was synthesized and then inserted between KpnI and NheI. The microplasminogen (μPg) was prepared through PCR, and inserted between Spe1 and XhoI. The flexible linker (GGGSG) was synthesized, and then inserted between Sa1I and SpeI, and finally the linker (GSEFVDGGGSGTA) was produced between the μPg and ferritin in the CLT-sFt-μPg structure. The sFt-μPg was constructed using the same method as above except for the insertion of the CLT peptide. The free μPg (free μPg) and the CLT-conjugated μPg were constructed using the same vector and restriction sites as above.

(25) 2. Characterization of CLT-sFt-μPg Double Chambered Nanocages (DCNCs)

(26) After expression and purification of proteins, the double chambered nanocage (DCNC) proteins (CLT-sFt-μPg and sFt-μPg) were analyzed using the dynamic light scattering (DLS) instrument (ELS-Z, Otzuka Electronics, Japan). The shape and size of the double chambered nanocages were observed using transmission electron microscopy (TEM).

(27) Each sample was diluted to 0.2 mg/mL, and applied to CF-200-Cu grids (Electron Microscopy Sciences), and washed three times. Thereafter, the sample was negatively stained with 2% uranyl acetate, and images were acquired using TEI Tecnai at the Korea Institute of Science and Technology.

(28) 3. Structure Modeling of CLT-sFt-μPg DCNC

(29) The structure of CLT-sFt-μPg single unit was modeled using MODELLER v9.12 on the basis of the crystal structures of human ferritin (PDB 2FG4) and microplasminogen (PDB 1QRZ). A total of 1,000 monomeric structures were produced, and assembled into CLT-sFt-μPg DCNC based on the wild-type ferritin cage structure (PDB 3A68) using PyMOL. At least 20 structures deviating from the standard were re-established using GROMACS, and a structure that showed no deviation from the standard and the lowest energy was selected as a model.

(30) 4. Clot Binding Analysis

(31) Clots were formed by adding CaCl.sub.2 (10 mM) and thrombin (0.5 U/mL) to fresh frozen plasma (FFP, 0.2 mL) and standing the mixture at 37° C. for 1 hour, and washed thoroughly using PBS. FITC-labeled proteins (1-4 μM) were added to on the clots, followed by incubation at 37° C. for 30 minutes. Thereafter, washing for removing the unbound substances was carried out. The fluorescence was monitored at an excitation wavelength of 488 nm and an emission wavelength of 520 nm using the SPECTA MAX BEMINI EM (MOLECULAR DEVIDES). For microscopic observation, the thin-layered clots were placed on the 1 mm-thick glass plates, and each protein was incubated at a concentration of 1.25 μM, washed with PBS, and observed under a fluorescence microscope. To verify that the activity of the peptides linked to both termini of the short ferritin fragment is not affected by the formation of the cage, equivalent molar concentrations of ferritin monomer and free protein were also incubated.

(32) 5. Clot Lysis Ability (Turbidity) Analysis

(33) The analysis was performed at room temperature in costar 96-well EIA/RIA plates (in triplicate) using the SUNRISE-BASIC reader (TECAN, Switzerland). Clots were generated by the same method as described above. For dissolution of the formed clots, each protein (7 μM) was added to the clots. Before clot dissolution, the microplasminogen or microplasminogen-fused peptides were activated by urokinase at 37° C. for 1 hour. Therefore, clot lysis was performed in the presence of urokinase.

(34) 6. Analysis of α2-Anti-Plasmin in Plasma and Investigation of Bleeding Side Effects

(35) Levels of α2-anti-plasmin in rodent were analyzed according to the previous reported method (Blood 97, 3086-3092, 2001). The activated CLT-sFt-μPn (7.9 mg/kg) and μPn (5 mg/kg) proteins were injected into via tail veins of ICR mice (6-8 week old: 18-26 g) (μPn: microplasmin). The same volume of saline was injected for control. 20 μL of animal blood was collected at the predetermined times (15 minutes, 60 minutes, and 120 minutes), and the plasma was prepared. To analyze the levels of α2-anti-plasmin, in vitro plasmin activity was measured before and after the mixing with rodent plasma.

(36) 10 μL of plasma was diluted using 420 μL of 0.05 M Tris-HCl buffer (pH 7.4), 100 mM NaCl, and 0.01% Tween 20, and 5 nM plasmin was added. After incubation for 10 seconds, 50 μL of 3 mM S2403 (Chromogenics, Antwerp, Belgium) was added to reaction samples, and the absorbance changes were measured at 405 nm. The absorbance changes were about 0.18 per minute in the buffer alone treatment group (i.e., 0% α2-anti-plasmin) and about 0.09 per minute in the plasma of the animals treated with saline (i.e., 100% α2-anti-plasmin). Calibration curves were made on the basis of the above results.

(37) In addition, after wounds were created in tail ends of ICR mice (6-8 week old: 18-26 g) administered with the activated CLT-sFt-μPn (7.9 mg/kg) and μPn (5 mg/kg) proteins, the time to stop bleeding was measured. Same volumes of saline and tPA were administered for control.

(38) 7. Arterial Thrombosis Model

(39) Male ICR (6-8 week old, 18-25 g) were housed in a pathogen-free environment with a temperature and humidity maintained. The mice were anesthetized, and the skin was incised to expose the right common carotid artery. The fascia was directly incised, and the right common carotid artery was partially exposed. Clots were induced by inserting a piece of filter paper sufficiently wet with FeCl.sub.3 (5%) under the right common carotid artery, and the inserted filter paper was removed after 3 minutes.

(40) Two minutes after occlusion by clot formation, 100 μL of 64.25 μM CLT-sFt-μPn, μPn, and CLT-μPn were injected via tail vein. The injected proteins were activated by incubation with urokinase for 1 hour (1:20), and the same amount of urokinase was independently injected as control. The carotid arteries of mice were perfusion fixed, stained hematoxylin and eosin (H/E), and retrieved for histological analysis. Twelve transversal histology sections of injured carotid arteries were evenly cut and subjected to H/E staining, and observed under VECTRA 3.0 (Perkinelmer).

(41) The occluded area was measured by inForm program (Perkinelmer), and normalized in % in relation to the total vessel lumen to quantify the degree of thrombosis of each section. The average of twelve sections for each mouse treated with each protein was floated.

(42) 8. Deep Vein Thrombosis Model

(43) Vein thrombosis animal models were prepared as reported in the prior art document (Thrombisis and haemostasis 105, 1060-1071, 2011). Briefly, SD rats were anesthetized, and the superior vena cava and the inferior vena cava were exposed to be segregated from other adjacent organs. Each end (3 cm) of the vena cava was loosely tied with 2-0 silk thread, and branched vessels were tightly ligated. Immediately, 20 UI of thrombin was injected through the tail vein.

(44) Thirty minutes after occlusion by clot formation, CLT-sFt-μPn (7.92 mg/kg) or μPn (5 mg/kg) was intravenously injected though the via tail vein. Each of the injected proteins was pre-activated by incubation with urokinase for 1 hour, and a same amount of urokinase was independently injected as control. After 60 minutes, the veins were segregated and stored in a Petri dish containing PBS. The thrombolytic activity was evaluated by immediately measuring the wet weight of the clots.

(45) <Results>

(46) 1. Manufacturing of Cage Nanoparticles of Fusion Protein and Characterization Thereof

(47) To develop a microplasmin-based thrombolytic agent, the present inventors designed a double-chambered nanocage (DCNC). As shown in FIG. 1A, multivalent clot-targeting (CLT) peptides and microplasmin proteins coexist on the surface of DCNC.

(48) The ferritin forms a nanoparticle, such as a cage, and various functional fractions may be chemically or genetically conjugated onto such a cage. The idea regarding the use of DCNC is that the peptide and protein payloads can offer double activities. These activities are augmented by binding activity of the ligands and do not impede each other's function.

(49) The CLT (CNAGESSKNC) peptide used in the present invention was identified by phage display that can recognize fibrin-fibronectin complexes in clots. The present inventors used a short fragment (sFt) of the human ferritin light chain, and produced such a short fragment by removing the fifth helix of the wild-type ferritin. The peptides and proteins loaded in the cage formed by short ferritin monomers did not impede each other's binding activity and physiological activity.

(50) The microplasminogen (μPg) can be activated into microplasmin (μPn) by the cleavage of Arg-Val residues through urokinase. The activated microplasmin is a two-chain disulfide-linked serine protease, and is homologous to trypsin with the classic catalytic triad of His, Asp, and Ser.

(51) The microplasmin is converted from microplasminogen by a plasmin activation enzyme, such as tPA or UPA, and has fibrin cleaving/dissolving activity. UPA cleaves between Arg580 and Val581 of the microplasminogen to convert to the microplasminogen into microplasmin including Val581 to the end residue. Since the stability of the microplasmin is lowered at the neutral pH, the proteins are produced and purified in a form of microplasminogen, and then activated by UPA before use in the present experiments.

(52) To predict orientations and display patterns of CLT peptides and microplasminogen protein payloads, a structure model of CLT-sFt-μPg sub unit was built by homologous modeling with MODELLER v9.12 (FIG. 1A). As shown in FIG. 1A, four copies of microplasminogen, each of which is fused to the C-terminus of sFt, are assembled into 4-fold symmetry and show a petal-like appearance, and six petals are dispersed on the surface of the nanocage. Twenty four CLT peptides, each of which is linked to the N-terminus of sFt, are exposed toward to the outside of the nanocage (FIG. 1B). The microplasminogen payloads are oriented in a direction that can retain the activation cleavage site (Arg-Val), and the catalytic triad amino acid residues remained outside of the nanocage and accessible thereto (FIG. 1A).

(53) The CLT-sFt-μPg and sFt-μPg DCNCs were purified, and differential light scattering (DLS) was performed to understand characteristics thereof (FIGS. 2A and 2B). The two types of DCNCs showed average sizes of 17.9 nm and 16.1 nm, respectively, which were similar to the predicted size from the prediction model. In other words, the results indicate that the modification for fusing the two peptides (CLT and microplasminogen) does not affect the overall cage characteristics.

(54) 2. Evaluation of Thrombolytic Ability of Fusion Peptide Nanocage

(55) To assess the efficacy of CLT-sFt-μPg DCNC as a thrombolytic agent, (i) the clot-binding ability of the CLT peptide, (ii) the thrombolytic ability of microplasminogen and microplasmin, and (iii) the systemic inactivation of anti-plasmin were evaluated.

(56) To evaluate these, the present inventors constructed μPg, CLT-conjugated microplasminogen (CLT-μPg) fusion protein, and luciferase-conjugated sFt(sFt-Luc) nanocages as controls (FIG. 3A). Clots, formed by the addition of CaCl.sub.2) and thrombin to fresh frozen plasma (FFP), were incubated with proteins labeled with fluorescent groups, followed by washing to remove the unbound fluorescent substance.

(57) For microscopic observation, thin-layered clots were formed on glass plates and the bound proteins were monitored. The CLT-conjugated products, i.e., CLT-sFt-μPg DCNC and CLT-μPg fusion protein were co-distributed with clots (FIG. 3B).

(58) The present inventors used equivalent molar concentrations of ferritin monomers and glass proteins to verify that the activities of the cage payloads (CLT and microplasminogen) were not affected by the formation of the cage. The binding ability of each construct was quantified by incubating the fluorescence-labeled nanocages and proteins with clots in 96-well plates.

(59) As a result, the binding ability of CLT-sFt-μPg DCNC was significantly higher than that of the CLT-μPg fusion protein, implying that the CLT peptides target clots with enhanced affinity due to the enhanced binding activity by the cage structure (FIGS. 3C and 3D).

(60) The CLT-sFt-μPg DCNC, μPg, CLT-μPg protein, and sFt-μPg were activated using urokinase, and then incubated with clots in 96-well plates. As shown in clot turbidity analysis results in FIG. 4A, the activated μPn loaded in the cage or the free form of μPn effectively dissolved the clots and showed higher activity compared with urokinase alone (FIG. 4A). The slight increase of turbidity in the saline group was due to the slow formation of clots during the incubation time.

(61) These results suggested that the μPn loaded in the cage or free form of μPn effectively dissolve clots. As for in vitro experiment, when the μPn loaded in the cage or free form of μPn were directly incubated with clots, CLT did not affect the lytic activity thereof. However, it could be confirmed through the following results that the clot-targeting ability by CLT is essential and very important in clot dissolution in vivo.

(62) 3. Confirmation of Stabilization Effect of Microplasmin by Nanocage and Bleeding Side Effects

(63) The present inventors investigated by monitoring the level of circulating α2-anti-plasmin whether the cage protein structure can shield the activated μPn from being degraded by anti-plasmin in the blood. It has been reported that intravenous plasmin or microplasmin are degraded respectively, to reduce the level of α2-anti-plasmin in the body. Therefore, the reduced level of α2-anti-plasmin in the body indicates the degradation of plasmin or microplasmin.

(64) In addition, the tPA used in the clinic may cause systemic somatic hemorrhage. Therefore, to investigate whether the microplasmin loaded in the cage has bleeding side effects, wounds were created in tail ends of mice, and then the time to stop bleeding was measured.

(65) As shown in FIG. 4B, the level of circulating α2-anti-plasmin decreased more slowly following intravenous administration of the activated CLT-sFt-μPn DCNC, compared with the administration of free form of μPn. This means that the nanocage structure shielded the μPn loaded in the cage from being degraded by anti-plasmin.

(66) As shown in FIG. 4C, the times to stop bleeding in the mice treated with CLT-sFt-μPn and μPn were similar to that in saline control, but the time to stop bleeding was prolonged in the mice treated with tPA. It was therefore confirmed that tPA may cause bleeding side effects but CLT-sFt-μPn had no bleeding effects like μPn. Therefore, the fusion protein of the present invention can be advantageously used as a thrombolytic agent without side effects.

(67) 4. Evaluation of In Vivo Thrombolytic Activity

(68) Arterial thrombi are different from venous thrombi with respect to causes, characteristics, and disease consequences. However, both types of thrombi may be life-threatening and thus need to be promptly removed.

(69) To investigate the in vivo effect of the CLT-sFt-μPg DCNC as a thrombolytic agent, CLT-sFt-μPg DCNC and other control substances were intravenously administered in arterial thrombus mouse models.

(70) As shown in FIG. 5A, as a result of observation of transverse histological sections of the right central carotid artery, the blood lump occluded the blood vessel when the blood vessel was exposed to 5% FeCl.sub.3. As a result of administration of the activated CLT-sFt-μPn DCNC, the blood circulation was very improved, but the activated μPn, CLT-μPn, sFt-μPn, and urokinase alone failed to dissolve the blood clot or showed insufficient effects. Compared with effects of tPA, which is the thrombolytic agent currently used in clinics, the blood circulation improving effect of CLT-sFt-μPn was significantly excellent.

(71) As shown in FIG. 5B, the injection of the activated CLT-sFt-μPn DCNC showed substantial thrombolytic/clot-bursting activity. The thrombolytic activity of the CLT-sFt-μPn DCNC was due to the accurate in vivo clot-targeting resulting from the presence of the CLT moiety. As shown in FIG. 3B, the targeting failure of CLT-μPn was determined to be due to the fact that free μPn lost activity thereof by anti-plasmin more easily than caged μPn. In addition, even when compared with tPA, the CLT-sFt-μPn had significantly excellent thrombolytic/clot-bursting activity.

(72) As a result of observation of ex vivo images of right and left carotid arteries, the CLT-sFt-μPg DCNC very specifically targeted a clot area in the right central carotid artery. Contrast to the in vitro binding results, the CLT-μPg fusion protein did not show an effect of targeting clots in the right central carotid artery like free μPg. This may be the reason why the thrombolytic activity of the CLT-μPg fusion protein was low.

(73) Next, the present inventors evaluated the activity of the CLT-sFt-μPg DCNC to dissolve thrombi in veins. An abnormal thrombus in a vein restricts the return of blood to the heart, and results in pain and swelling. Deep vein thrombosis is a type of thrombi that are formed in a major vein of the leg. When such a thrombus separates, circulates, and blocks the heart and lung blood vessels, it causes an acute pulmonary embolism.

(74) As shown in FIG. 5C, the present inventors surgically block the inferior vena cava of the rat to form a prominent clot in the corresponding region. The rats treated with activated CLT-sFt-μPn DCNC developed smaller and lower-weight clots compared with the rats treated with saline or free μPn (FIG. 5D).

(75) As set forth above, the CLT-sFt-μPn DCNC according to the present invention is a novel plasmin-based thrombolytic nanocage, and has an effect of targeting a thrombus site, low susceptibility to inhibitors present in the circulatory systems, and pharmaceutical activity to strongly destruct both arterial and venous thrombi.

INDUSTRIAL APPLICABILITY

(76) The CLT-sFt-μPn DCNC according to the present invention is a novel plasmin-based thrombolytic nanocage, and has an effect of targeting a thrombus site, low susceptibility to inhibitors present in the circulatory systems, and pharmaceutical activity to strongly destruct both arterial and venous thrombi, and thus has superior industrial applicability.