Cardiomyocyte surface-modified with collagen-hybridized peptide and use thereof

Abstract

The present invention relates to a cardiomyocyte surface-modified with a collagen-hybridized peptide and a use thereof. A cardiomyocyte surface-modified with a collagen-hybridized protein according to the present invention can have large molecules of the collagen-hybridized protein effectively inserted into the cell surface by using click chemistry, and the optimal conditions thereof were determined. In addition, it was found that the cardiomyocyte can be specifically attached to denatured collagen due to being surface-modified with the collagen-hybridized protein. In addition, it was found that, when transplanted into a myocardial infarction animal model, the cardiomyocyte increases cardiac function, attaches to areas of denatured collagen in cardiac tissue, thereby regenerating fibrotic cardiomyocytes, and inhibits cardiac remodeling, thereby providing recovery from cardiac damage after myocardial infarction.

Description

Cardiac muscle cells surface-modified with collagen hybridization peptides and uses thereof

[0001] The present invention relates to a cardiac muscle cell surface-modified with a collagen hybridization peptide and the use thereof.

[0002] Ischemia refers to a condition of reduced blood supply to body organs, tissues, or areas caused by the constriction or occlusion of blood vessels. Even if repurfusion occurs after ischemia, nerve cells are damaged, leading to various sequelae. Such ischemia is often associated with coronary artery disease, cardiovascular disease, angina, headaches, or other vascular symptoms, and ultimately leads to irreversible damage, namely necrosis of cells and tissues.

[0003] Ischemic diseases such as myocardial infarction, arrhythmia, and failure, caused by cell damage and functional decline during ischemia and reperfusion, have high morbidity and mortality rates and are difficult to cure; consequently, intensive basic and clinical research has been conducted over the past 50 years. Since ischemia and reperfusion injury involve various physiological mechanisms—including metabolism, immune responses, changes in ion homeostasis, and oxygen free radicals—research is being conducted in diverse fields, such as immunomodulatory substances, apoptosis-related substances, and ion channel regulators. While active efforts have been made to develop therapeutic agents targeting new targets and surgical procedures alongside mechanistic studies, technologies capable of protecting myocardial cells from ischemia and reperfusion have not yet been clinically commercialized. Therefore, there is an urgent need for the development of preventive and therapeutic agents for ischemic heart disease, or cardioprotective agents, that can slow the progression of ischemia-induced myocardial cell damage and mitigate reperfusion injury.

[0004] Meanwhile, acute myocardial infarction (AMI), also known as cardiac arrest, is a condition in which blood flow to one of the coronary arteries is blocked, potentially damaging the heart muscle or leading to death. Billions of cells, amounting to up to 25% of the total left ventricular mass, are damaged within hours of the onset of myocardial infarction. Since the heart has limited regenerative capacity after birth, it is known that approximately 25% of myocardial infarction patients suffer from left ventricular systolic dysfunction and are at risk of progressive cardiac remodeling. Treatment for acute myocardial infarction generally involves restoring blood flow to the affected area of ​​the heart as quickly as possible through procedures such as percutaneous coronary intervention or coronary bypass graft surgery, but this is merely a method to slow progression to end-stage heart failure and is not the optimal treatment method; furthermore, drug treatment is mainly aimed at managing symptoms and neurohormonal activity, and there is a lack of treatments to fundamentally restore cardiomyocytes (CM).

[0005] Ongoing research aimed at overcoming the therapeutic limitations of myocardial infarction focuses on discovering new treatments to restore damaged cardiac tissue and improve patients after the infarction. Accordingly, methods are being investigated to activate various cell types that differentiate cardiovascular cells, including stem cells and progenitor cells, and to induce their migration to damaged areas. Another approach being investigated involves promoting the proliferation of resident cardiomyocytes in vivo or transplanting new cardiomyocytes to replace necrotic tissue. Cardiomyocytes are muscle cells that make up the heart and contract and relax to supply blood throughout the body. Human pluripotent stem cells (hPSC-CM) currently provide an infinite supply of differentiated human cardiomyocytes. As it has become known that hPSC-CM can promote myocardial regeneration in damaged cardiac tissue, it has been reported that transplanting hPSC-CM improves cardiac contractility, reduces scar formation, and promotes angiogenesis in infarcted cardiac tissue.

[0006] However, even when hPSC-CM is transplanted for cardiac regeneration, the transplantation and survival rates of hPSC-CM are low in the environment of the infarct site, which includes ischemia, inflammation, and fibrosis. Therefore, to increase transplantation and cell survival rates, it is necessary to optimize the transplantation conditions of hPSC-CM by reconstructing the regenerative microenvironment. Currently, to effectively transplant hPSC-CM, a cardiac patch containing 3D stem cells has been developed. When implanted into the infarcted heart site, it increases the transplantation and survival rates of hPSC-CM, thereby enhancing cardiac regeneration after myocardial infarction. However, since a thoracotomy is required to implant the cardiac patch, it can place a burden on the patient. Therefore, there is a need for a method to increase the transplantation and survival rates of single myocardial cells after transplantation.

[0007] Accordingly, the inventors completed the present invention by producing surface-modified cardiomyocytes using bioorthogonal chemistry with click chemistry and confirming the cardiac regeneration effect after myocardial infarction.

[0008] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of myocardial infarction comprising cardiomyocytes surface-modified with collagen hybridized peptide (CHP) as an active ingredient.

[0009] Another objective of the present invention is to provide a cell therapy agent for the prevention or treatment of myocardial infarction comprising cardiomyocytes surface-modified with collagen hybridized peptide (CHP) as an active ingredient.

[0010] Another objective of the present invention is the step of inserting lipids into myocardial cells;

[0011] A step of combining a collagen hybridization peptide with trans-cyclooctene (TCO);

[0012] A step of mixing the above lipid-inserted cardiomyocytes and the collagen hybridization peptide combined with TCO; and

[0013] The present invention provides a method for preparing a cardiac cell surface-modified with a collagen hybridization peptide, comprising the step of inducing bioorthogonal chemistry of the above lipids and TCOs.

[0014] Another objective of the present invention is to provide a method for preventing or treating myocardial infarction comprising the step of administering the pharmaceutical composition or the cell therapy agent to an individual.

[0015] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of myocardial infarction comprising cardiomyocytes surface-modified with collagen hybridized peptide (CHP) as an active ingredient.

[0016] In addition, the present invention provides a cell therapy agent for the prevention or treatment of myocardial infarction comprising cardiomyocytes surface-modified with collagen hybridized peptide (CHP) as an active ingredient.

[0017] In addition, the present invention comprises the step of inserting lipids into myocardial cells;

[0018] A step of combining a collagen hybridization peptide with trans-cyclooctene (TCO);

[0019] A step of mixing the above lipid-inserted cardiomyocytes and the collagen hybridization peptide combined with TCO; and

[0020] A method for preparing a cardiac cell surface-modified with a collagen hybridization peptide is provided, comprising the step of inducing bioorthogonal chemistry of the above lipids and TCOs.

[0021] In addition, the present invention provides a method for preventing or treating myocardial infarction comprising the step of administering the above pharmaceutical composition or the above cell therapy agent to an individual.

[0022] Cardiac cells surface-modified with the collagen hybridization protein of the present invention can effectively insert large molecules of the collagen hybridization protein onto the cell surface using click chemistry, and the optimal conditions for this were identified. Furthermore, it was confirmed that surface-modified with the collagen hybridization protein can specifically attach to denatured collagen. Additionally, when transplanted into an animal model of myocardial infarction, it enhances cardiac function, attaches to denatured collagen regions within cardiac tissue to regenerate fibrotic cardiomyocytes, and inhibits cardiac remodeling, thereby recovering cardiac damage after myocardial infarction; thus, it can be usefully applied in related industries.

[0023] Figure 1 illustrates the overall experimental purpose of the present invention.

[0024] Figure 2 is a schematic diagram illustrating the optimization process of hPSC-CM fabrication according to the present invention.

[0025] FIGS. 3 to 5 are figures confirming the optimal solvent and optimal lipid treatment method for the production of hPSC-CM according to the present invention (Fig. 3: confirmation of optimal solvent, Fig. 4: confirmation of lipid treatment time, Fig. 5: confirmation of lipid treatment concentration).

[0026] Figures 6 and 7 illustrate the optimal TCO processing method for the fabrication of hPSC-CM according to the present invention (Figure 6: verification of optimal processing time, Figure 7: verification of optimal processing rate).

[0027] Figures 8 and 9 show the cell viability according to the production of the optimized hPSC-CM of the present invention (Figure 8: cell viability according to lipid treatment, Figure 9: cell viability according to hPSC-CM treatment).

[0028] Figures 10 to 13 show the optimized bioorthogonal chemistry of the present invention confirmed in the H9C2 cell line.

[0029] Figures 14 to 17 show the optimized bioorthogonal chemistry of the present invention confirmed in hPSC-EC cells.

[0030] Figures 18 to 21 show the optimized bioorthogonal chemistry of the present invention confirmed in MSC cells.

[0031] FIGS. 22 to 27 are figures confirming the usefulness of the bioorthogonal chemistry-based two-step cell surface modification method of the present invention.

[0032] Fig. 22: Schematic illustration of the differences between Step 1 and Step 2 cell surface modification methods

[0033] Fig. 23: Confirmation of ligand presentation efficiency of hPSC-CM

[0034] Fig. 24: Comparison of cell counts within positive gates in flow cytometry

[0035] Fig. 25: Quantification of average fluorescence intensity

[0036] Fig. 26: Confocal microscopy analysis results

[0037] Fig. 27: Results of fluorescence intensity comparison

[0038] Figures 28 to 32 show the adhesion of modified collagen of CHP and hPSC-mCM in vitro.

[0039] Fig. 28: Comparison of native collagen and denatured collagen structures

[0040] Fig. 29: Confirmation of CHP's specific attachment to denatured collagen

[0041] Fig. 30: Confirmation of CHP adhesion fluorescence intensity

[0042] Fig. 31: Confirmation of specific attachment of denatured collagen in hPSC-mCM

[0043] Fig. 32: Quantification of the number of hPSC-mCM cells attached to a collagen gel

[0044] Figure 33 illustrates the process of confirming the adhesion ability of CHP-mCM in heart tissue.

[0045] Figures 34 to 36 show the adhesion ability of CHP in heart tissue from which cells have been removed.

[0046] Fig. 34: Schematic representation of the method for verifying CHP adhesion ability

[0047] Fig. 35: Confirmation of fluorescence intensity according to CHP attachment

[0048] Fig. 36: Quantification of fluorescence intensity

[0049] Figures 37 to 40 show the adhesion ability of hPSC-mCM in cell-removed heart tissue, confirmed by fluorescence analysis and confocal microscopy.

[0050] Fig. 37: Confirmation of fluorescence expression

[0051] Fig. 38: Quantification of fluorescence expression

[0052] Fig. 39: Confocal microscopy analysis results

[0053] Fig. 40: Quantification of confocal microscopy results

[0054] Figures 41 and 42 show collagen denaturation over time after myocardial infarction confirmed by confocal microscopy (Figure 41: confirmation and quantification of cTnT expression, Figure 42: confirmation of type 1 collagen and denatured collagen).

[0055] FIGS. 43 and 44 are diagrams showing the cardiac function in an animal model of myocardial infarction following the transplantation of the hPSC-mCM of the present invention by echocardiography (Fig. 43: M-mode analysis results, Fig. 44: quantification of indices related to left ventricular myocardial function).

[0056] Figure 45 is a figure quantifying the results of echocardiography measurements in an animal model of myocardial infarction following the transplantation of the hPSC-mCM of the present invention.

[0057] FIGS. 46 and 47 are figures quantifying hemodynamic indices and pressure-volume relationships in an animal model of myocardial infarction following the transplantation of hPSC-mCM of the present invention (A: quantification of hemodynamic pressure and volume, B: quantification of pressure-volume relationship).

[0058] Figure 48 is a figure quantifying indices related to cardiac output and stroke volume in an animal model of myocardial infarction following the transplantation of hPSC-mCM of the present invention.

[0059] FIGS. 49 and 50 are figures confirming histological changes in the heart in an animal model of myocardial infarction following transplantation of the hPSC-mCM of the present invention by MT staining (Fig. 49: staining result, Fig. 50: quantification of staining result).

[0060] Figures 51 and 52 show the expression of cTnT and CHP in an animal model of myocardial infarction following the transplantation of the hPSC-mCM of the present invention using a confocal microscope (Figure 51: analysis results, Figure 52: quantification of analysis results).

[0061] Figures 53 and 54 are confocal microscopy analyses of cTnT and CM in the borderline and infarct zones in an animal model of myocardial infarction following the transplantation of hPSC-mCM of the present invention (Figure 53: analysis results, Figure 54: quantification of analysis results).

[0062] FIGS. 55 to 57 show the engraftment ability of hPSC-CM in an animal model of myocardial infarction following the transplantation of hPSC-mCM of the present invention by immunofluorescence staining (Fig. 55: iPSC-CM staining result, Fig. 56: cTnT staining result, Fig. 57: quantification of staining result).

[0063] Figures 58 and 59 show the expression of CX43 and total cTnT in an animal model of myocardial infarction following transplantation of the hPSC-mCM of the present invention by immunofluorescence staining (Figure 58: staining result, Figure 59: quantification of staining result).

[0064] The terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Therefore, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as “comprising” a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0065] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise noted.

[0066]

[0067] The present invention provides a pharmaceutical composition for the prevention or treatment of myocardial infarction comprising cardiomyocytes surface-modified with collagen hybridized peptide (CHP) as an active ingredient.

[0068] The term “prevention” as used in this invention refers to any act of suppressing the symptoms of a specific disease or delaying its progression through the administration of the composition of this invention.

[0069] The term “treatment” as used in this invention refers to any act of improving or beneficially altering the symptoms of a specific disease through the administration of the composition of this invention.

[0070] The pharmaceutical composition of the present invention may additionally include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete or incomplete adjuvant may be further included to increase the effect.

[0071] The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited to these, but may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0072] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external formulations, suppositories, or sterile injectable solutions, each according to conventional methods.

[0073] When formulating, the product may be prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and such solid dosage forms may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the active ingredient. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Water-insoluble solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Bases for suppositories may include Witepsol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc.

[0074] The pharmaceutical composition according to the present invention may be administered to an individual by various routes. Any mode of administration may be anticipated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0075] The dosage of the pharmaceutical composition according to the present invention is selected by taking into consideration the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and preferably, it is included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not appear, and if it exceeds 5,000 μg / ml, it may exhibit toxicity to the human body.

[0076] The "collagen hybridized peptide (CHP)" of the present invention is a peptide synthesized to form a triple helix structure by mimicking the characteristic sequence of natural collagen. CHP is a peptide with increased hybridization properties with collagen chains, possessing a repeat sequence of glycine (G)-proline (P)-hydroxyproline (O), which is the amino acid triplet with the highest folding propensity for the triple helix structure found in all GXY repeat units of the collagen sequence. Collagen is a major component of the extracellular matrix present in the tissues and organs of almost all mammals; while the synthesis and degradation of collagen are regulated in tissue development and homeostasis processes, excessive collagen remodeling is associated with various pathological conditions. Collagen forms a triple helix structure, and all 28 collagen subtypes share a supersecondary structure as a basic structural motif in which three protein strands are intertwined in a triple helix structure stabilized by hydrogen bonds; this triple helix structure is found only in collagen in the human body. During the collagen remodeling process, when collagen is degraded by actions such as collagen-lytic protease, the fragmented collagen triple helix loses thermal stability even within the body temperature range and spontaneously unfolds to form denatured collagen, and CHP forms a triple helix structure from this denatured collagen and crossbreds.

[0077] The collagen hybridization peptide of the present invention is a peptide having the amino acid sequence NH2-GGGGfOGfOGfOGfOGfOGfOGfOGfOGfOGfO-COOH, wherein "f" in the sequence is (2S, 4S)-4-fluoroproline and "O" is L-hydroxyproline, and both are proline variants, so the amino acid sequence of SEQ ID NO. 1 can be represented as GGGGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP.

[0078] According to one embodiment of the present invention, the collagen hybridization peptide may be inserted into a cardiomyocyte by bioorthogonal chemistry, and the bioorthogonal chemistry may be performed with tetrazine or trans-cyclooctene (TCO).

[0079] Meanwhile, a "click reaction" refers to a reaction in which different chemical functional groups simply combine; it is a chemical reaction that occurs easily without the application of heat or complex catalysts. Click reactions exhibit high selectivity and react rapidly, yielding products with high yields without unnecessary byproducts. A well-known representative click reaction is the "Cu-catalyzed Azide-Alkyne Cycloaddition (CuAAC)."

[0080] The "Bioorthogonal chemistry" of the present invention is a type of click reaction that can be utilized in vivo. The above CuAAC reaction cannot be utilized in vivo because copper binds to various substances in the body and exerts toxic effects. Accordingly, by using an unstable alkyne instead of a stable alkyne structure, it is possible to bind to a desired molecule in vivo without using a copper catalyst.

[0081] According to one embodiment of the present invention, the tetrazine may be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-tetrazine](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-tetrazine], DSPE-PEG2k-Tz).

[0082] According to one embodiment of the present invention, the DSPE-PEG2k-Tz may be inserted into the cell membrane of a myocardial cell.

[0083] According to one embodiment of the present invention, the TCO may be connected to the CHP.

[0084] According to one embodiment of the present invention, the collagen hybridization peptide TCO may be combined and linked to the DSPE-PEG2k-Tz of the myocardial cell membrane via bioorthogonal chemistry.

[0085] According to one embodiment of the present invention, the surface-modified myocardial cells may specifically bind to denatured collagen.

[0086] According to one embodiment of the present invention, the surface-modified myocardial cells may increase cardiac function after myocardial infarction, and increasing cardiac function may increase the left ventricular ejection fraction and reduce the remodeling of the cardiac damaged area.

[0087] According to one embodiment of the present invention, reducing the remodeling may be reducing the left ventricular internal diameter end-diastole and reducing fibrosis within the myocardial tissue.

[0088] According to one embodiment of the present invention, the surface-modified myocardial cells may have an increased engraftment or transplantation rate within myocardial tissue compared to a control group.

[0089] According to one embodiment of the present invention, the composition may reduce degenerated collagen precipitation in heart tissue.

[0090]

[0091] In addition, the present invention provides a cell therapy agent for the prevention or treatment of myocardial infarction comprising cardiomyocytes surface-modified with collagen hybridized peptide (CHP) as an active ingredient.

[0092]

[0093] In addition, the present invention comprises the step of inserting lipids into myocardial cells;

[0094] A step of combining a collagen hybridization peptide with trans-cyclooctene (TCO);

[0095] A step of mixing the above lipid-inserted cardiomyocytes and the collagen hybridization peptide combined with TCO; and

[0096] A method for preparing a cardiac cell surface-modified with a collagen hybridization peptide is provided, comprising the step of inducing bioorthogonal chemistry of the above lipids and TCOs.

[0097]

[0098] In addition, the present invention provides a method for preventing or treating myocardial infarction comprising the step of administering the above pharmaceutical composition or the above cell therapy agent to an individual.

[0099] The term "individual" as used in this invention refers to a subject requiring a method for the prevention, control, or treatment of a disease, and may be used without limitation and includes humans, dogs, monkeys, cats, rodents, e.g., mice, genetically modified mice, etc. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0100] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0101]

[0102] <Experimental Example 1> Preparation for Confirmation of Myocardial Cell Surface Modification and Cardiac Regeneration Effects

[0103] <1-1> Preparation of Reagents and Materials

[0104] DSPE-PEG2k-Tz (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-tetrazine]) and TCO-FITC (trans-cyclooctene-fluorescein isothiocyanate) were purchased from Ruixi Biotech (China). DSPE-PEG2k-FITC (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000-fluorescein isothiocyanate]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000-fluorescein isothiocyanate]) was purchased from Biopharma PEG Scientific Inc (Watertown, MA, USA). Amine-modified collagen hybridization peptide (CHP) was purchased from 3helix (Salt Lake City, UT) (NH2-GGGGfOGfOGfOGfOGfOGfOGfOGfOGfOGfO-COOH; f = (2S, 4S)-4-fluoroproline, O = L-hydroxyproline; SEQ ID NO. 1: GGGGPPGPPGPPGPPGPPGPPGPPGPPGPP), and TCO was conjugated to amine-CHP using TCO-N-hydroxysuccinimide (Click Chemistry Tools; Scottsdale, AZ, USA). Collagen and Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, MO, USA).Collagen antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and Calcein Deep Red and AM Ester were purchased from AAT Bioquest (Sunnyvale, CA, USA).

[0105]

[0106] <1-2> Cell Surface Modification

[0107] Cardiomyocytes surface-modified with the collagen hybridized peptide (CHP) of the present invention were prepared. Specifically, a two-step cell surface modification was performed. DSPE-PEG2k-Tz was inserted into the cell membrane of human pluripotent stem cells (hPSC-CM) differentiated from human pluripotent stem cells (cardiomyocytes differentiated by treating human induced pluripotent stem cell line CMC11 cells with CHIR-99021 (CT99021); GSK-3α and GSK-3β inhibitors (Sigma) for 2 days, followed by the addition of WNT-C59; WNT inhibitor (Selleckchem) for 2 days), and any uninserted lipids were removed by washing with PBS (step 1). Subsequently, the cells were detached from the culture plate and reacted with TCO-CHP for 10 minutes (step 2). Afterward, the hPSC-CM surface-modified with CHP (CHP-mCM) was collected by centrifugation. In addition, cells were prepared using TCO-FITC instead of TCO-CHP for fluorescence imaging and flow cytometry analysis. As a control, DSPE-PEG2k-FITC was treated to hPSC-CM for 30 minutes to remove residual lipids by washing with PBS, and the lipids inserted into the hPSC-CM were analyzed by flow cytometry.

[0108]

[0109] <1-3> In Vitro Cell Adhesion Test

[0110] To evaluate the binding of CHP and denatured collagen, CHP-FITC was applied to native or denatured collagen gels for 30 minutes. Afterward, both gels were washed with PBS, and fluorescence images were obtained using an Amersham ImageQuant 800 system (Cytiva, USA). To evaluate cell adhesion, CHP-mCM was applied to each of the two gels, stained with Hoechst 33342, and incubated for 4 hours. Unbound cells were removed, and fluorescence images were obtained using a confocal laser scanning microscope (LSM800, Carl Zeiss).

[0111] To confirm binding with cardiac tissue, 6 to 8-week-old mice were humanely sacrificed, and cardiac tissue was obtained. The obtained cardiac tissue was treated with trypsin, and cells were removed using Triton X-100. Subsequently, collagen within the cardiac tissue was denatured using SDS solution. The denatured cardiac tissue was treated with CHP-FITC for 30 minutes, and the binding of CHP to the denatured cardiac extracellular matrix was confirmed by observing the fluorescence signal. After confirming CHP binding, CHP-mCM was applied to the denatured cardiac tissue and incubated for 4 hours, after which the tissue was washed with PBS. Subsequently, cells were stained with calcein stain, and the fluorescence signal of hPSC-CM in the denatured cardiac tissue was stained. Collagen was stained with an anti-collagen antibody for confocal fluorescence imaging.

[0112]

[0113] <1-4> Myocardial Infarction Animal Model and CHP-Surface Modified CM Transplantation

[0114] Myocardial infarction induction and CHP-mCM transplantation were performed with the approval of the Institutional Animal Care and Use Committee of the Catholic University of Korea (Approval No.: CUMC-2023-0234-01) and in compliance with NIH guidelines and the European Parliament Directive 2010 / 63 / EU on the protection of animal research. Specifically, Fischer 344 rats (male, 8 weeks old, body weight 160–180 g; Central Lab (SLC), Seoul, Korea) were anesthetized with 2% isoflurane (Hana Pharm, Seongnam, Korea), endotracheally intubated using an 18-gauge intravenous catheter (Korea Vaccine, Seoul, Korea), and mechanically ventilated with a VentElite® (Harvard Apparatus, USA). To suppress the risk of infection during surgery, gentamicin at a concentration of 5 mg / kg and the analgesic ketoprofen at a concentration of 5 mg / kg were administered intramuscularly (IM). After shaving the chest wall, a left thoracotomy was performed, and myocardial ischemia was induced by ligating the left anterior descending artery (LAD) for 1 hour using 7-0 Florene sutures (Ethicon, Inc., Somerville, NJ, USA). After 1 hour, the knot was removed to reperfuse the LAD, the incision was aseptically sutured, and the chest was disinfected. One week after the ischemia / reperfusion (I / R) injury, a baseline echocardiogram (Affiniti 50G, Philips, Andover, MA, USA) was performed prior to CHP-mCM transplantation. Mice were anesthetized with 2% isoflurane, and under mechanical ventilation, the left thoracotomy site was reopened to transplant CHP-mCM and surface-modified CM (nCM) at a density of 1.0 × 10⁶ 6The cells were transplanted after being suspended in 50 μl of PBS at a cellular concentration. Afterward, the chest was aseptically sutured and disinfected, and standard immunosuppressants including azathioprine (2 mg / kg, Korea United Pharm Inc., Seoul, Korea), cyclosporine A (5 mg / kg, CKD Pharm, Seoul, Korea), and methylprednisolone (5 mg / kg, Hanlim Pharm, Seoul, Korea) were administered to all animal models.

[0115]

[0116] <1-5> Continuous transthoracic echocardiography

[0117] To evaluate the function of the heart with I / R injury, serial transthoracic echocardiography was performed. Specifically, mice were anesthetized with 2% isoflurane during the procedure, and cardiac function was evaluated using an Affiniti50G ultrasound system equipped with a 15 MHz L15-7io linear transducer (Philips, Andover, MA, USA). Echocardiography was performed one week after the induction of I / R injury, and additional echocardiography was performed at 1, 2, 4, and 6 weeks after CHP-mCM and nCM transplantation. The echocardiographer did not disclose information regarding each group throughout the experiment, and left ventricular (LV) systolic function was evaluated using ejection fraction (EF) and fractional shortening (FS) calculated using Equations 1 and 2 below.

[0118]

[0119]

[0120]

[0121] In the above mathematical formulas 1 and 2, LVEDV represents the left ventricular end-diastolic volume, LVESV represents the left ventricular end-systolic volume, LVEDD represents the left ventricular end-diastolic diameter, and LVESD represents the left ventricular end-systolic diameter.

[0122]

[0123] <1-6> Pressure-Volume Loop Analysis

[0124] At the end of the experiment at week 6, hemodynamics were measured using pressure-volume loop analysis (PV loop analysis) before mouse sacrifice. Specifically, mice in each group were anesthetized with 2% isoflurane, and thoracotomy was performed without inducing hemorrhage. Subsequently, the LV apex of the heart was punctured using a 26-gauge needle, and a 2F conduction catheter (SPR-838; Millar Instruments, Houston, TX, USA) was inserted into the LV. PV parameters were continuously recorded using a PV conduction system (MPVS Ultra; emka TECHNOLOGIES, Paris, France) and a digital transducer (PowerLab 16 / 35; ADInstruments, Chalgrove, UK). Based on the recorded values, stroke volume (SV), cardiac output (CO), maximal rate of pressure changes during systole (dP / dtmax), minimal rate of pressure changes during diastole (dP / dtmin), maximum volume at end-diastole (Vmax), and maximum pressure at end-diastole (Pmax) were quantified. Using a needle holder, intrinsic cardiac contractile load, including the slopes of the end-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure-volume relationship (EDPVR), was measured independently. After that, 50 μl of hypertonic saline (20% NaCl) was injected into the left jugular vein to measure hemodynamics, and then parallel conductivity was evaluated.Blood collected from the LV apex using heparin was placed into a cuvette, and the conductivity signal was converted into volume using a catheter, and the absolute blood volume of the mouse heart was confirmed through the correction of parallel conductivity and cuvette conductivity.

[0125]

[0126] <1-7> Immunohistochemical staining

[0127] At the end of the experiment, mice were humanely sacrificed and cardiac tissues were obtained. The cardiac tissues were then fixed with 4% paraformaldehyde, paraffin blocks were prepared, and the tissues were sectioned. After paraffin removal and rehydration, antigens were recovered using a desilvered lung chamber containing a target recovery solution. The sections were then incubated with diluted primary antibodies for 18 hours. Mouse anti-total cardiac troponin T (#ab8295, 1:200, Abcam), rabbit anti-human cardiac troponin T (#ab45932, 1:300, Abcam), and rabbit anti-connexin-43 (#ab11370, 1:300, Abcam) were used as primary antibodies. After the reaction, the sections were washed four times with PBS and incubated with secondary antibodies at room temperature in a dark room for 90 minutes. Anti-mouse Alexa Fluor 488 (#A11001, 1:200, Thermo Fisher Scientific) and anti-rabbit Alexa Fluor 647 (#A31573, 1:500, Thermo Fisher Scientific) were used as secondary antibodies. After the reaction was complete, sections were washed with PBS and stained with an anti-fade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vectashield, H-1200-10) for nuclear staining. Denatured collagen was stained using 5-carboxyfluorescein-conjugated CHP (#FLU300, 20 μM, 3HELIX, Salt Lake City, UT, USA). Stained images were obtained using a confocal microscope (LSM900, Carl Zeiss, Jena, Germany).

[0128]

[0129] <1-8> Histological Analysis

[0130] The cardiac tissue sections prepared in Experimental Examples 1-7 above were stained with Masson's trichrome staining to identify fibrotic sites and surviving myocardium of the damaged heart. Specifically, the paraffin sections were incubated in a drying oven at 37°C for 18 hours, after which the paraffin was removed and the sections were rehydrated. Subsequently, the sections were refixed in Bouin solution at 56°C for 1 hour and 30 minutes. Afterward, the slides were washed with water for 20 minutes. The sections were then reacted with Weigert's iron hematoxylin solution at room temperature for 15 minutes, followed by staining with Biebrich's scarlet-acid fuchsin solution at room temperature for 20 minutes. Afterward, the sections were counterstained with aniline blue for 15 minutes, and then treated with 1% acetic acid and reacted at room temperature for 2 minutes. Cardiac section imaging was performed using a slide scanner (Pannoramic MIDI), and fibrotic areas indicated in blue and viable myocardium indicated in red were quantified using ImageJ software.

[0131]

[0132] <1-9> Quantitative Image Analysis of Cardiac Cell Transplantation and Engraftment

[0133] Total CM was quantified based on the number of cardiac troponin T (cTnT)-positive cells, and the area of ​​denatured collagen was quantified using CHP-positive regions. The presence of transplanted human CM and lateralized Connexin 43 (CX43) was quantified by analyzing count values ​​in immunofluorescence images of red fluorescence protein (RFP)-positive and human cTnT-positive cells with lateralized CX43-positive junctions, respectively. Quantitative analysis of immunofluorescence images was performed in three randomly selected regions per tissue, and measurements were given in millimeters per gram ( / mm²).2 It was expressed as a party.

[0134]

[0135] <1-10> Statistical Analysis

[0136] Statistical analysis was performed using GraphPad Prism 9 (GraphPad, La Jolla, CA, USA), and statistical significance was determined by one-way analysis of variance (ANOVA) using Tukey's multiple comparison test for each analysis.

[0137]

[0138] <Example 1> Lipid Insertion and Click Chemistry Optimization for Cell Surface Modification

[0139] It was confirmed whether the lipid insertion and click chemistry-based two-step cell surface modification method of the present invention increases cell surface modification ability compared to direct one-step surface modification. Specifically, the two-step cell surface modification method was optimized using hPSC-CM and flow cytometry. Surface modification ability was evaluated by changing the treatment solution, lipid treatment time, lipid concentration, TCO treatment time, and TCO treatment ratio (Fig. 2). DSPE-PEG2k-FITC was used as the model lipid for the one-step surface modification method in the lipid insertion optimization, while DSPE-PEG2k-Tz and TCO-FITC were used as models for the two-step cell surface modification method of the present invention. As a result, it was confirmed that the optimal treatment solution was PBS containing 10% serum (Fig. 3). In addition, it was confirmed that the amount of lipids inserted into hPSC-CM did not significantly increase even with an increase in lipid treatment time, and that lipid insertion actually decreased when treated for more than 4 hours; thus, the optimal lipid treatment time was selected as 30 minutes (Fig. 4). It was confirmed that the optimal lipid concentration was 1 mg / ml, at which no lipid-induced toxicity occurred (Fig. 5). After optimizing the lipid insertion conditions, the click chemistry reaction was optimized. When the TCO treatment time was set to 10 minutes, more than 80% of the hPSC-CMs showed a positive reaction (Fig. 6). Subsequently, the optimized TCO treatment concentration was checked, and when compared to the lipid concentration, it was confirmed that hPSC-CMs treated with 0.01 equivalents of TCO-TIFC relative to the lipid concentration reached a 100% level of cell surface modification and exhibited a fluorescence signal more than 7 times higher compared to hPSC-CMs treated with 0.1 equivalents of TCO-TIFC without lipid insertion (Fig. 7). In addition, the cell viability of lipid-containing hPSC-CM did not show toxicity up to 1 mg / ml and was confirmed to be similar to that of surface-unmodified hPSC-CM (Figs. 8 and 9).The surface modification method described above could be completed within one hour and was confirmed to be applicable to various cell types, including rat embryonic cardiac myoblast cell line H9C2, human pluripotent stem cell-derived endothelial cells, and mesenchymal stem cells, and it was confirmed that the optimized conditions also showed a similar pattern regardless of the cell type (Figs. 10 to 21).

[0140]

[0141] <Example 2> Confirmation of the usefulness of click chemistry-based two-step cell surface modification

[0142] The utility of the click chemistry-based two-step cell surface modification of the present invention was confirmed. Specifically, the ability to directly insert the lipid conjugate in the first step into the cell membrane is reduced due to the large size of molecules such as CHP. Specifically, as confirmed in various studies, the molecular weight of antibodies is approximately 150 kDa and the molecular weight of fibronectin is approximately 500 kDa, which is larger than the optimized PEG length of 2 kDa; furthermore, the molecular weight of CHP of the present invention is 3 kDa, which is larger than the optimized PEG. It is known that molecules with high molecular weights interfere with lipid insertion, and it has been reported that increasing the optimized PEG of 2 kDa to 10 kDa reduces lipid insertion efficiency to less than 1%. Accordingly, in order to confirm the utility of the two-step cell surface modification method using click chemistry-based bioorthogonal chemistry in the present invention, DSPE-PEG5k-FITC, a model lipid for the first step, and DSPE-PEG2k-Tz and TCO-FITC, model lipids for the two-step cell surface modification were used. As a result, it was confirmed that the two-step surface modification method exhibited a binding efficiency 4.66 times higher compared to the one-step surface modification method (Figs. 23 and 24). In addition, when comparing the one-step surface modification method and the two-step surface modification method in a cell suspension, it was confirmed that the two-step surface modification method had an 8.65 times higher surface modification ability in flow cell analysis compared to the one-step surface modification method. Furthermore, it was confirmed that the two-step surface modification method of the present invention exhibited uniformity, with more than 99% of hPSC-CM distributed within the positive gate, whereas only 20% of the one-step surface modification method was distributed within the positive gate (Fig. 25). In the two-step surface modification method, it was confirmed that the attached dye was mainly located on the cell surface in CLSM, and evaluated by scanning the fluorescent signal lines in the fluorescence image (Figs. 26 and 27).From the above results, it was confirmed that the click chemistry-based two-step cell surface modification method of the present invention can efficiently introduce large molecular weight molecules to the cell surface.

[0143]

[0144] <Example 3> Confirmation of tissue adhesion of CHP-mCM (in vitro)

[0145] The tissue adhesion of the CHP-mCM of the present invention was confirmed in vitro. Specifically, undenatured native collagen and denatured collagen gels were prepared, each gel was treated with a CHP-chromosome conjugate, and washed to evaluate the binding ability of CHP (Fig. 28). As a result, it was confirmed that CHP specifically binds to denatured collagen (Figs. 29 and 30). Subsequently, the adhesion of CHP-mCM to collagen gels was confirmed. While there was no significant difference in the adhesion of nCM and CHP-mCM to native collagen, it was confirmed that the adhesion of CHP-mCM to denatured collagen gel increased, resulting in a 2.82-fold increase in the cell count (Figs. 31 and 32).

[0146] Subsequently, to confirm the attachment of CHP-mCM to denatured collagen in cardiac tissue, mouse cardiac tissue was decellularized and collagen within the extracellular matrix was denatured (Fig. 33). The CHP-chromosome conjugate was treated to the denatured cardiac tissue to confirm the adhesive ability of CHP to denatured collagen within the cardiac ECM (Fig. 34). It was confirmed that the CHP-chromosome conjugate exhibited a 6.5-fold higher fluorescence signal in the denatured cardiac tissue compared to the undenatured control cardiac tissue (Figs. 35 and 36). Furthermore, strong fluorescence expression was observed as the attachment of CHP-mCM to the denatured cardiac tissue increased (Figs. 37 and 38), and CLSM confirmed that the number of attached CHP-mCM cells in the denatured cardiac tissue was 19.55-fold higher compared to nCM, thereby confirming that the adhesive ability of CHP-mCM to denatured collagen increased (Figs. 39 and 40).

[0147]

[0148] <Example 4> Confirmation of Improvement in Cardiac Function After Myocardial Infarction Following CHP-mCM Transplantation

[0149] We investigated whether the transplantation of the CHP-mCM of the present invention could improve cardiac function after myocardial infarction. To determine whether the accumulation of degenerated collagen is induced in the infarcted heart and to determine the timing of CHP-mCM transplantation, the amount of degenerated collagen in the infarcted heart was evaluated first. Targeting collagen chains using 5-carboxyploid olesane-conjugated CHP, it was confirmed that degenerated collagen gradually accumulates starting from 3 days after the induction of myocardial infarction and increases on the 7th day after I / R injury (Figs. 41 and 42). Subsequently, based on the expression pattern of degenerated collagen after myocardial infarction, 7 days after MI was determined as the timing for CHP-mCM transplantation. Subsequently, CHP-mCM was implanted on the 7th day after MI, and cardiac function was evaluated using continuous echocardiography at weeks 1, 2, 4, and 6. As a result, it was confirmed that at week 6, the LVEF in the group implanted with CHP-mCM was significantly increased compared to the control group (nCM). In addition, the SWT was thicker, and the left ventricular internal diameter end-diastole (LVID) was significantly reduced in the CHP-mCM group compared to the control group (nCM), confirming that the implantation of CHP-mCM improved cardiac function and also reduced the remodeling of negative myocardial tissue in I / R-impaired hearts (Figs. 43 to 45).

[0150] Subsequently, to verify the role of CHP-mCM in cardiac regeneration, a 6-week PV catheter was directly inserted into the LV after transplantation, and PV loop analysis was performed. The results confirmed that load-dependent hemodynamic parameters, including stroke volume and cardiac output, increased in the CHP-mCM group (Figs. 46 and 48). Load-independent parameters, including ESPVR and EDPVR, were also improved (Figs. 47 and 48). These results were statistically significant compared to the nCM group, confirming that CHP-mCM restored systolic and diastolic function in the infarcted heart regardless of the preload volume status.

[0151]

[0152] <Example 5> Confirmation of Effects of CHP Surface Modification

[0153] We confirmed whether the CHP surface modification of the present invention increased the transplantation rate of CM cells and the regeneration of myocardial damage. Specifically, mice in each group were humanely sacrificed at the end of the experiment, and cardiac tissues were obtained, sectioned, and histologically analyzed. Masson's trichrome staining confirmed that, compared to the control nCM group, the CHP-mCM group showed a significant reduction in the fibrilized area of ​​the LV wall and a significant increase in viable myocardial portion within the infarct area (Figs. 49 and 50). Quantifying the ratio of the total number of cTnT-positive CMs to the transplanted CHP-mCMs in the infarcted heart revealed that the total number of cTnT-positive CMs in the CHP-mCM group significantly increased (Fig. 51), and that degenerated collagen deposition in the infarct area was reduced compared to the nCM group (Figs. 51 to 54). In addition, it was confirmed that the RFP-labeled CHP-mCM group was integrated into host cTnT-positive CM compared to the nCM group (Figs. 55 to 57). From the above results, it was confirmed that CHP-mCM increased the graft rate in denatured collagen sites through CHP binding to denatured collagen and was integrated into viable myocardial tissues in fibrotic areas. Furthermore, staining for CX43, a protein involved in gap junction formation via cell signaling in CM, revealed that lateralized CX43 was significantly reduced in the CHP-mCM group compared to the nCM group, confirming that CHP-mCM was well integrated into myocardial cells (Figs. 58 and 59).

[0154]

[0155] Therefore, the cardiac muscle cells surface-modified with the collagen hybridization protein of the present invention can effectively insert large molecules of the collagen hybridization protein onto the cell surface using click chemistry, and the optimal conditions for this were identified. Furthermore, it was confirmed that the cells, surface-modified with the collagen hybridization protein, can specifically attach to denatured collagen. Additionally, it was confirmed that when transplanted into an animal model of myocardial infarction, they enhance cardiac function, attach to denatured collagen regions within cardiac tissue to regenerate fibrotic cardiac cells, inhibit cardiac remodeling, and restore cardiac damage after myocardial infarction.

Claims

1. A pharmaceutical composition for the prevention or treatment of myocardial infarction comprising cardiomyocytes surface-modified with collagen hybridized peptide (CHP) as an active ingredient.

2. In Paragraph 1, A composition in which the above collagen hybridization peptide is inserted into cardiomyocytes via bioorthogonal chemistry.

3. In Paragraph 2, A composition in which the above bioorthogonal chemistry is performed with tetrazine or trans-cyclooctene (TCO).

4. In Paragraph 3, A composition in which the above tetrazine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-tetrazine](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-tetrazine], DSPE-PEG2k-Tz).

5. In Paragraph 4, The above DSPE-PEG2k-Tz is a composition that is inserted into the cell membrane of a myocardial cell.

6. In Paragraph 3, The above TCO is a composition connected to CHP.

7. In Paragraph 1, A composition that binds to the above collagen hybridization peptide TCO and is bioorthogonally linked to DSPE-PEG2k-Tz of the myocardial cell membrane.

8. In Paragraph 1, A composition in which the surface-modified myocardial cells specifically bind to denatured collagen.

9. In Paragraph 1, A composition in which the surface-modified myocardial cells increase cardiac function after myocardial infarction.

10. In Paragraph 9, A composition in which increasing the above cardiac function increases the left ventricular ejection fraction.

11. In Paragraph 9, A composition that increases the above-mentioned heart function, which reduces the remodeling of the heart damaged area.

12. In Paragraph 11, A composition in which reducing the above remodeling reduces the left ventricular internal diameter end-diastole.

13. In Paragraph 11, A composition that reduces the above remodeling, which reduces fibrosis within myocardial tissue.

14. In Paragraph 1, A composition in which the surface-modified myocardial cells have an increased engraftment or transplantation rate within myocardial tissue compared to a control group.

15. In Paragraph 1, The above composition is a composition that reduces degenerated collagen precipitation in heart tissue.

16. A cell therapy product for the prevention or treatment of myocardial infarction comprising cardiomyocytes surface-modified with collagen hybridized peptide (CHP) as an active ingredient.

17. Step of inserting lipids into myocardial cells; A step of combining a collagen hybridization peptide with trans-cyclooctene (TCO); A step of mixing the above lipid-inserted cardiomyocytes and the collagen hybridization peptide combined with TCO; and A method for preparing surface-modified cardiomyocytes with collagen hybridization peptides, comprising the step of inducing bioorthogonal chemistry of the above lipids and TCOs.

18. In Paragraph 17, A method in which the above lipid is tetrazine.

19. In Paragraph 18, A method in which the above tetrazine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-tetrazine](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000-tetrazine], DSPE-PEG2k-Tz).

20. A method for preventing or treating myocardial infarction comprising the step of administering the pharmaceutical composition of claim 1 or the cell therapy agent of claim 16 to an individual.

Images