Polydopamine and antibody coated medical devices

Abstract

To provide medical devices with coatings, which are artificial heart valves, vascular stent grafts, or synthetic grafts.SOLUTION: The present disclosure provides a medical device or substrate coated with polydopamine which is further linked to ligands such as antibodies and / or antibody fragments. The polydopamine coating and the ligands may be linked through a linker such as an organic polymer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is incorporated herein by reference in its entirety. ,223 (filed April 13, 2017) and No. 62 / 645,606 (filed April 13, 2018) The application claims priority from the application filed on March 20th.

[0002] The present invention relates to medical devices, such as intravascular devices, coated with polydopamine and antibodies. Regarding vessels. [Background technology]

[0003] The immobilization of biomolecules is of great interest in both biology and physics. An active area of research in this field is the development of bioactive coatings for intravascular devices. These devices (coronary stents, vascular grafts, etc.) are associated with significant mortality and morbidity. Coronary artery disease (CAD) and peripheral artery disease (PAD), which are known to cause used to treat 1 Treatment options include risk factor modification and the introduction of novel drug therapies. Advances have significantly reduced the incidence of atherosclerotic vascular disease and improved its prognosis; Surgical vascular bypass grafting and percutaneous transluminal coronary angioplasty with endovascular stent placement Peritoneal coronary angioplasty (PTCA) continues to be among the most common procedures performed annually in North America. 2 These revascularization techniques are routinely used, but unfortunately, the use of stents Long-term success is limited by restenosis at the treatment site and late stent thrombosis. On the other hand, the short- and mid-term success of synthetic grafts used to treat PAD depends on their thrombus formation. Limited by composition.

[0004] Intra-arterial stents are available in a wide range of materials (e.g., 316L stainless steel (SS), tantalum, Nitinol, Cobalt-Chromium (CoCr) alloy, Platinum-Iridium, Polymer, etc. The cylindrical mesh is used for stent implantation in a process called intimal hyperplasia. Restenosis, the reduction in luminal expansion after intervention, remains one of the most important problems facing interventionalists. Reduces early restenosis through the local release of cytotoxic compounds into the vessel wall Drug-eluting stents designed to prevent complications such as late thrombosis and late restenosis are It was found that drug-eluting stents induce smooth muscle cells (SMCs), which are the cause of restenosis. C) not only inhibits the growth of comfrey covering the stent, a process critical to healing. It was soon recognized that it also inhibits the formation of a pervasive endothelial cell (EC) layer.

[0005] Expanded polytetrafluoroethylene (ePTFE) is the material most frequently used for arterial reconstruction. The high flow rate of the large-diameter vascular grafts allowed for 85% of patients with minimal adjunctive medical therapy. Provides a long-term (>10-year) patency rate of ~95% 3 However, small-diameter artificial blood vessels The successful development of prostheses (<5 mm) was primarily driven by the high thrombogenicity of ePTFE. This continues to be a challenge due to the shortened patency caused by this interface. This may be further perpetuated by the lack of EC contact inhibition in the situ, leading to EC hyperplasia. be 4 Failure of synthetic grafts to fully endothelialize in humans is a common cause of failed arterial reconstruction. Continuous thrombosis at the surface of the graft, ultimately contributing to the development of the underlying myointimal hyperplasia causes disease events 5~7In fact, fewer than 50% of small-diameter femoral-popliteal grafts survive for 5 days after implantation. Remains patent at 1 year 8 .

[0006] Failure of complete surface re-endothelialization of prosthetic endovascular materials is common in humans, but A dense EC covering is common in other mammalian species. 9 Established endothelium on the prosthetic graft The source of the cells is thought to be from capillary invasion or ingrowth from the edge of an adjacent artery. It was given 10 However, this paradigm has recently been called into question. In implanted highly porous ePTFE grafts, capillary ingrowth occurs from the outside of the graft. It has been shown that it rarely exceeds half the distance to the lumen 11 Rather, The main source of the sparse endothelial lining of prosthetic implants in patients is the "fallout" It has been shown that this can occur from the circulating blood through a process called "healing." There are 12 In a subsequent study by Shi et al., fallout ECs were derived from bone marrow. It is coming 13、14 , which have been further demonstrated to be circulating endothelial progenitor cells (EPCs) in the blood. Ta.

[0007] The arterial endothelium is a dynamic organ, with its relaxation and contraction as well as the functions of fibrinolysis, thrombus formation, and platelet activation / inhibition, thereby maintaining vascular homeostasis. This formation of active organs is observed in blood vessels after stent implantation and in the ECs may provide favorable biological properties in the prosthetic graft. This disrupts SMC proliferation by impairing the stent surface and prosthetic graft material, leaving them passivated. and prevent thrombosis 15~17 Confluent endothelial lining on implanted vascular devices Recognition of the importance of perforation has led to EC as a means to improve their long-term patency. This prompted research into the seeding of vascular stents and grafts. Herring, 1978 Since the first introduction of EC seeding in 2000, many groups have contributed to the development of this technology. However, the success of each group has been limited. 18~60 Self-EC is the best way to serve your organization. It is generally agreed that the limited availability of resources for the self-EC The availability and tedious process of seeding and implantation can affect the surface of the prosthesis. coupled with a failure to achieve a predictable confluent monolayer of cells on Furthermore, the structure and biochemical environment of the venous bed, arterial bed, microvascular bed, and macrovascular bed are all different. Therefore, the arrangement of ECs from one bed to another is crucial for cell performance. The best approach for re-endothelialization of the material is to Accelerate the process of fallout healing by accelerating the attraction of EPCs to the It will be 61 . 245μm 2 Based on an average EC area of 4100 cells / mm 2 density of Capturing EPCs with endothelial cells provides complete coverage of the material surface, which is essential for the development of vascular prostheses. Leads to effective endothelialization 62 .

[0008] We designed, developed, and tested an EPC-capturing intracoronary stent. 63~65 .child The stent is made of polymer dextromethorphan embedded with a mouse monoclonal anti-human CD34 antibody. The trans-coating is used to capture EPCs and enhance the natural endothelialization process. The xylan coating technology has been proven to be effective in capturing CD34+ cells. Similar to our dextran coating, other antibodies for the capture of specific cell types have been used. Immobilization strategies have been somewhat successful. Unfortunately, they are often limited in scope and These compounds are material-specific, suffer from loss of biological activity, and require labor-intensive chemistry. , we have developed a novel method for the immobilization of biologically active molecules that can be effectively applied to a wide range of substrates. The aim is to develop a universal method.

[0009] Valves are essential for the normal physiological function of the cardiovascular system. For example, natural heart valves They ensure one-way blood flow from one heart chamber to another. Natural heart or venous valves can be compromised by various pathologies. Some conditions require complete surgical replacement of the natural valve with a valve prosthesis. Prosthetic heart valves are devices implanted in the hearts of patients with valvular heart disease. .

[0010] Despite significant improvements in prosthetic valve design and surgical procedures over the past few decades, valve replacement remains a challenging task. Instead, the prognosis for patients undergoing valve replacement is significantly affected by the prosthetic valve. The hemodynamics, durability, and thrombogenicity of

[0011] Dopamine (DA, short for 3,4-dihydroxyphenethylamine) regulates the brain and body Catecholamines and phenethylamines play several important roles in the body. Polydopamine (PDA) is a synthetic dopamine-derived organic chemical. Polydopamine is a melanin polymer that adheres to many types of surfaces at a slightly basic pH. However, there is little information regarding the mechanism of formation. A fundamental understanding is still lacking. Lynge et al., Polydopam ine-a nature-inspired polymer coating fo r biomedical science,Nanoscale,2011,3:49 16.

[0012] An ideal primer coating would be one that can be universally applied to any substrate. In this regard, simple immersion of the substrate in a dilute aqueous solution of dopamine buffered to alkaline pH Since the discovery that polydopamine films can be spontaneously deposited on substrates, The use of polydopamine in the treatment of rheumatoid arthritis has attracted considerable interest. Messersmith et al. al (Science, 2007, 318, 426-430) reported that polydopamine The coating is compatible with metals, metal oxides, ceramics, synthetic polymers, and a wide range of other hydrophilic materials. It can be formed on virtually any type of substrate surface, including both hydrophilic and hydrophobic materials. Polydopamine coating is a novel method for the application of synthetic polymers or biomolecules to surfaces. For example, International Publication No. 2011 / 0 No. 05258 discloses the addition of amine functional groups to a polydopamine coating to provide a hydrophilic outer layer. discloses the attachment of functionalized polyethylene glycol (PEG-NH2).

[0013] In terms of durability, the coating is designed to withstand the slow erosion of the coating material. and / or the coating is peeled off from the surface of the substrate. Therefore, one way to increase the durability of a coating is to The goal is to strengthen the bond between the coating and the surface of the substrate. This is especially important when Treat the surface to be coated with a primer to achieve better adhesion between the This can be achieved by: [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2011 / 005258 [Non-patent literature]

[0015] [Non-Patent Document 1] Lynge et al.,Polydopamine-a nature-inspired polymer coating for biomedical science,Nanoscale,2011,3:4916 [Non-patent document 2] Messersmith et al(Science,2007,318,426-430) Summary of the Invention [Problem to be solved by the invention]

[0016] The present disclosure provides a medical device having a coating, the coating comprising (i) polydopa (ii) a polyether derivative; and (iii) an antibody and / or antibody fragment. Polydopamine is covalently bound to the polyether derivative, and the polyether derivative acts as an anti- and / or antibody fragments. do.

[0017] The antibody and / or antibody fragment is specific for a cell surface antigen of an endothelial progenitor cell or an endothelial cell. can bind to

[0018] The present disclosure provides a medical device having a coating, the coating comprising (i) polydopa (ii) a polyether derivative; and (iii) an antibody and / or antibody fragment. Polydopamine is covalently bound to the polyether derivative, and the polyether derivative acts as an anti- and the antibody and / or antibody fragment is covalently bound to an endothelial progenitor antibody. Medical devices having coatings that specifically bind to cell surface antigens of cells or endothelial cells to provide.

[0019] 1. A prosthetic valve having a coating, the coating comprising: (i) polydopamine; i) a polyether derivative; and (iii) an antibody and / or antibody fragment, The polyether derivative is covalently bound to the antibody and / or or an antibody fragment, and the prosthetic valve is a prosthetic heart valve or a prosthetic venous valve. Prosthetic valves having fins are also encompassed by the present disclosure.

[0020] Non-limiting examples of cell surface antigens include CD34, CD133, CDw90, CD117, H LA-DR, VEGFR-1, VEGFR-2, VEGFR-3, Muc-18(CD1 46), Thy-1, Thy-2, CD130, CD30, stem cell antigen (Sca-1), Stem cell factor 1 (SCF / c-Kit ligand), Tie-1, Tie-2, VE-cadherin Lin, P1H12, TEK, CD31, Ang-1, Ang-2, HAD-DR, CD4 5, CD105, CD14, von Willebrand factor (vWF), and E-selectin Includes:

[0021] Polyether derivatives include polyethylene glycol (PEG), polyethylene glycol (PEG) derivatives, polypropylene glycol (PPG), polypropylene glycol ( PPG) derivatives, or combinations thereof.

[0022] PEG is from about 200 daltons to about 20,000 daltons, from about 200 daltons to about 5,0 00 Daltons, about 200 Daltons to about 1,000 Daltons, about 200 Daltons to about 350 It may have an average molecular weight in the range of Daltons.

[0023] Medical devices include stents, artificial heart valves, vascular prosthetic filters, catheters, and pacemakers. -, vascular grafts, synthetic grafts, pacemaker leads, defibrillators, patent foramen ovale (PF O) Septal closure devices, vascular clips, vascular aneurysm occluders, hemodialysis grafts, hemodialysis devices catheters, atrioventricular shunts, aortic aneurysm graft devices or components, artificial veins Valves, shunts, wires, sensors, sutures, vascular anastomosis clips, indwelling venous or arterial catheters The catheter may be a catheter, a vascular sheath, or a drug delivery port.

[0024] The medical device is a prosthetic valve, such as a prosthetic aortic valve, prosthetic pulmonary valve, prosthetic mitral valve, or prosthetic tricuspid valve. It may be a heart valve or an artificial venous valve.

[0025] The medical device may comprise a metal (such as stainless steel), an alloy, and / or a polymer. Polymers include polytetrafluoroethylene (PTFE), Dacron, polyurethane, and poly be a biocompatible polymer such as propylene, or combinations or derivatives thereof; can be done.

[0026] The coating may or may not further comprise a pharmaceutical agent. The agent inhibits smooth muscle cell migration and / or proliferation. In another embodiment, the pharmaceutical agent , a vasodilator.

[0027] Non-limiting examples of pharmaceutical agents include paclitaxel, rapamycin, rapamycin derivatives, cyclosporine ... lorlimus, everolimus, tacrolimus, biolimus, biolimus A-9, or any of its This includes combinations of these.

[0028] The antibodies and / or antibody fragments can be monoclonal or polyclonal. The antibody and / or antibody fragment may be a humanized antibody or antibody fragment, or a chimeric antibody. or antibody fragments. The antibody and / or antibody fragment may be Fab, F(a) or F(b) or F(c) or F(d ... b') 2, or a single chain Fv (scFv).

[0029] In one embodiment, the antibodies and / or antibody fragments specifically bind to different cell surface antigens. do.

[0030] The antibodies and / or antibody fragments of the medical device may act as antibodies when the medical device is implanted in a subject. Endothelial progenitor cells and / or endothelial cells can be captured in vivo.

[0031] The present disclosure provides a method for treating or preventing vascular disease, comprising implanting the medical device in a patient. The present invention provides a method for treating or preventing vascular disease, comprising the step of implanting a vascular

[0032] Vascular disease may be atherosclerosis, restenosis, thrombosis, and / or vascular occlusion. It is possible. [Brief explanation of the drawings]

[0033] [Figure 1A] Figure 1 shows different antibody immobilization techniques. Figure 1A: Non-oriented immobilization using dextran coating. Figure 1B: Non-oriented immobilization using amine coupling. Both 1A and 1B can result in buried antigen-binding regions. Figure 1C: Oriented immobilization of an antibody to a PEG-modified surface via the modified Fc region of the antibody. The antigen-binding site remains available for immunobinding. [Figure 1B] Figure 1 shows different antibody immobilization techniques. Figure 1A: Non-oriented immobilization using dextran coating. Figure 1B: Non-oriented immobilization using amine coupling. Both 1A and 1B can result in buried antigen-binding regions. Figure 1C: Oriented immobilization of an antibody to a PEG-modified surface via the modified Fc region of the antibody. The antigen-binding site remains available for immunobinding. [Figure 1C] Figure 1 shows different antibody immobilization techniques. Figure 1A: Non-oriented immobilization using dextran coating. Figure 1B: Non-oriented immobilization using amine coupling. Both 1A and 1B can result in buried antigen-binding regions. Figure 1C: Oriented immobilization of an antibody to a PEG-modified surface via the modified Fc region of the antibody. The antigen-binding site remains available for immunobinding. [Figure 2A] The scheme for forming this coating is shown in Figure 2A. Polydopamine deposition—the basis for coating. Figure 2B. Linker layer polyethylene glycol (PEG) deposition—the basis for oriented antibody coating. Figure 2C. Oriented antibody coating. [Figure 2B] The scheme for forming this coating is shown in Figure 2A. Polydopamine deposition—the basis for coating. Figure 2B. Linker layer polyethylene glycol (PEG) deposition—the basis for oriented antibody coating. Figure 2C. Oriented antibody coating. [Figure 2C] The scheme for forming this coating is shown in Figure 2A. Polydopamine deposition—the basis for coating. Figure 2B. Linker layer polyethylene glycol (PEG) deposition—the basis for oriented antibody coating. Figure 2C. Oriented antibody coating. [Figure 3]FIG. 1 illustrates the general structure of an embodiment of the present coating. [Figure 4A] FIG. 1 shows exemplary structures and polymer configurations that polydopamine can form from dopamine by oxidative self-polymerization in a basic environment, such as occurs when coating a substrate / medical device (e.g., 316L stainless steel (316S), cobalt chromium (CoCr), ePTFE, or pericardium). [Figure 4B] FIG. 1 shows exemplary structures and polymer configurations that polydopamine can form from dopamine by oxidative self-polymerization in a basic environment, such as occurs when coating a substrate / medical device (e.g., 316L stainless steel (316S), cobalt chromium (CoCr), ePTFE, or pericardium). [Figure 5] 1 is an exemplary reaction scheme for oxidative activation of an antibody or antibody fragment for subsequent coupling to a nucleophilic presenting surface. [Figure 6-1] 1 is an exemplary reaction scheme (oxidation method) for coating a substrate / endovascular material / medical device (e.g., 316L stainless steel (316L SS), cobalt-chromium (CoCr), ePTFE, or pericardium) with an antibody (e.g., anti-CD34 antibody) that is conjugated to polydopamine via an intermediate PEG linker. As an example, PEG is shown to be conjugated to polydopamine via Michael addition. [Figure 6-2] 1 is an exemplary reaction scheme (oxidation method) for coating a substrate / endovascular material / medical device (e.g., 316L stainless steel (316L SS), cobalt-chromium (CoCr), ePTFE, or pericardium) with an antibody (e.g., anti-CD34 antibody) that is conjugated to polydopamine via an intermediate PEG linker. As an example, PEG is shown to be conjugated to polydopamine via Michael addition. [Figure 6-3]1 is an exemplary reaction scheme (oxidation method) for coating a substrate / endovascular material / medical device (e.g., 316L stainless steel (316L SS), cobalt-chromium (CoCr), ePTFE, or pericardium) with an antibody (e.g., anti-CD34 antibody) that is conjugated to polydopamine via an intermediate PEG linker. As an example, PEG is shown to be conjugated to polydopamine via Michael addition. [Figure 7-1] 1 is an exemplary reaction scheme for coating a substrate / endovascular material / medical device (e.g., 316L stainless steel (316L SS), cobalt-chromium (CoCr), ePTFE, or pericardium) with an antibody (e.g., an anti-CD34 antibody) that is conjugated to polydopamine via an intermediate PEG linker. As examples, PEG is shown to be conjugated to polydopamine via a Michael addition or Schiff base reaction. [Figure 7-2] 1 is an exemplary reaction scheme for coating a substrate / endovascular material / medical device (e.g., 316L stainless steel (316L SS), cobalt-chromium (CoCr), ePTFE, or pericardium) with an antibody (e.g., an anti-CD34 antibody) that is conjugated to polydopamine via an intermediate PEG linker. As examples, PEG is shown to be conjugated to polydopamine via a Michael addition or Schiff base reaction. [Figure 8A] 8A-8C are exemplary reaction schemes (enzymatic methods) for coating substrates / intravascular materials / medical devices with antibodies linked to polydopamine via an intermediate PEG linker. Figure 8A: Polydopamine-coated substrates are reacted with amino-PEG-dibenzocyclooctyne (DBCO). Figure 8B: Functionalization of antibodies (e.g., at the Fc region of the antibody) to create DBCO-reactive moieties. Step 1 shows the removal of terminal galactose residues, and step 2 shows the incorporation of GalNAz. See Zeglis et al., Chem. 2013, 24(6), 1057-1067; Qu et al., Adv. Healthc. Mater. 2014, 3(1), 30-35. Figure 8C: Reaction of functionalized antibodies with PEG linkers. [Figure 8B]8A-8C are exemplary reaction schemes (enzymatic methods) for coating substrates / intravascular materials / medical devices with antibodies linked to polydopamine via an intermediate PEG linker. Figure 8A: Polydopamine-coated substrates are reacted with amino-PEG-dibenzocyclooctyne (DBCO). Figure 8B: Functionalization of antibodies (e.g., at the Fc region of the antibody) to create DBCO-reactive moieties. Step 1 shows the removal of terminal galactose residues, and step 2 shows the incorporation of GalNAz. See Zeglis et al., Chem. 2013, 24(6), 1057-1067; Qu et al., Adv. Healthc. Mater. 2014, 3(1), 30-35. Figure 8C: Reaction of functionalized antibodies with PEG linkers. [Figure 8C] 8A-8C are exemplary reaction schemes (enzymatic methods) for coating substrates / intravascular materials / medical devices with antibodies linked to polydopamine via an intermediate PEG linker. Figure 8A: Polydopamine-coated substrates are reacted with amino-PEG-dibenzocyclooctyne (DBCO). Figure 8B: Functionalization of antibodies (e.g., at the Fc region of the antibody) to create DBCO-reactive moieties. Step 1 shows the removal of terminal galactose residues, and step 2 shows the incorporation of GalNAz. See Zeglis et al., Chem. 2013, 24(6), 1057-1067; Qu et al., Adv. Healthc. Mater. 2014, 3(1), 30-35. Figure 8C: Reaction of functionalized antibodies with PEG linkers. [Figure 9] 1 is a graph showing the fluorescence intensity of the binding of an azide-functionalized fluorescent probe (carboxyrhodamine 110-azide) on a dibenzocyclooctyne (DBCO)-functionalized substrate (e.g., a disk). "Bare": a bare metal disk of CoCr that is neither treated nor coated. "Bare + DBCO": a bare metal disk coated only with DBCO, but not with polydopamine. "PDOP": a bare metal disk coated with polydopamine. "PDOP + DBCO": a bare metal disk coated with polydopamine and then DBCO. [Figure 10]This photograph shows the capture of CD34-expressing Kg1a cells ("positive") on a 316L SS coronary stent coated with anti-CD34 antibody (BioLegend, catalog #343602) via an intermediate amino-dPEG8-t-boc-hydrazide linker attached to polydopamine formed by oxidative self-polymerization of dopamine. The coating was blocked with bovine serum albumin (BSA) before incubation with the cells. Bound cells were visualized by nuclear dye Sytox Green staining on a confocal microscope. Control cells were CHO cells ("negative") that do not express CD34. [Figure 11] This photograph shows the capture of CD34-expressing Kg1a cells ("positive") on cobalt chromium (CoCr) discs coated with anti-CD34 antibody (BioLegend, #343602) via an intermediate amino-dPEG8-t-boc-hydrazide linker attached to polydopamine formed by oxidative self-polymerization of dopamine. The coating was blocked with bovine serum albumin (BSA) before incubation with the cells. Bound cells were visualized by nuclear dye Sytox Green staining on a confocal microscope. Control cells were CHO cells ("negative") that do not express CD34. [Figure 12] This photograph shows the capture of CD34-expressing Kg1a cells ("positive") on a medical-grade expanded polytetrafluoroethylene (ePTFE) endograft coated with anti-CD34 antibody (BioLegend, #343602) via an intermediate amino-dPEG8-t-boc-hydrazide linker attached to polydopamine formed by oxidative self-polymerization of dopamine. The coating was blocked with bovine serum albumin (BSA) before incubation with the cells. Bound cells were visualized by nuclear dye Sytox Green staining on a confocal microscope. Control cells were CHO cells ("negative") that do not express CD34. [Figure 13]Photographs showing the stability of CD34+ or CD34- cells bound to ePTFE coated with anti-CD34 antibodies. The ePTFE substrates were placed in PBS for 12 days and then used for cell binding. The bound cells were stained with a fluorescent dye and observed under a confocal microscope. [Figure 14] This photograph shows the capture of CD34-expressing Kg1a cells ("CD34+ cells") on bovine pericardium coated with anti-CD34 antibody (BioLegend, #343602) via an intermediate amino-dPEG8-t-boc-hydrazide linker attached to polydopamine formed by oxidative self-polymerization of dopamine. The coating was blocked with bovine serum albumin (BSA) before incubation with the cells. Bound cells were visualized by nuclear dye Sytox Green staining on a confocal microscope. Control cells were CHO cells that do not express CD34 ("CD34- cells"). [Figure 15]

[0023] Figure 1 shows a graph showing a cytotoxicity assay for anti-H-2Kk antibody-coated ePTFE grafts. The coated grafts were incubated with CHO H-2Kk(+) cells for 1, 2, or 3 days. They were then fixed and imaged by fluorescence microscopy. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present disclosure provides a method for the preparation of antibodies which are further linked to a ligand / biomolecule such as an antibody and / or antibody fragment. melanin, melanin-like polymers, synthetic versions of melanin, or aromatic catechols Coating with a polymer (e.g., polydopamine or a polymer of a dopamine analog) The polydopamine coating and the ligand are The linker may be an organic polymer / oligomer or the like. ) polydopamine, (ii) organic polymers (e.g., polyethylene glycol (PEG) and other organic polymers / oligomers described herein), and (iii) antibody and / or antibody fragment-coated medical devices (e.g., Polydopamine is an organic polymer / oligomer that The organic polymer / oligomer may be covalently attached to the antibody and / or antibody fragment. Antibodies and / or antibody fragments, such as anti-CD34 antibodies, may be covalently attached to the pro-endothelial It can specifically bind to cell surface antigens / molecules of endothelial cells or endothelial progenitor cells (EPCs). and / or antibody fragments induce endothelial progenitor cells in vivo when the medical device is implanted in a subject. and / or capture endothelial cells. The medical device may also include a pharmaceutical or therapeutic agent.

[0035] The present disclosure provides a method for producing melanin-containing polymers, including: (i) melanin, melanin-like polymers, synthetic versions of melanin, or Aromatic catechol polymers (e.g., polydopamine or polymers of dopamine analogs) ), (ii) organic polymers (e.g., polyethylene glycol (PEG) and other polymers) ter derivatives, and other organic polymers / oligomers described herein), and (iii ) a pharmaceutical coated with a ligand / biomolecule (e.g., antibody and / or antibody fragment) melanin, melanin-like polymers, synthetic versions of melanin, or Aromatic catechol polymers (e.g., polydopamine or polymers of dopamine analogs) ) may be covalently attached to an organic polymer / oligomer, may be covalently bound to a ligand / biomolecule (e.g., antibody and / or antibody fragment) Ligands / biomolecules (e.g., antibodies and / or antibody fragments) can be used to bind to endothelial progenitor cells or or endothelial cells.

[0036] The coatings may be applicable to a wide range of substrates / materials, are biocompatible, and have a wide Offers facile chemistry and broad reactivity to a wide range of ligands / biomolecules. The biomolecules may be attached to the coating in an oriented manner. It also has long-term chemical stability.

[0037] In one embodiment, the polydopamine coating comprises a salt on the surface of the medical device or substrate. via oxidative self-polymerization of dopamine under basic conditions (e.g., slightly basic conditions) A polyethylene glycol (PEG) linker is then applied, which is Binding to the polydopamine coating at one end and the Fc fragment of an antibody or antibody fragment at the other end and combine.

[0038] The coating of the medical device may further comprise an antibody, an antibody fragment, or a combination thereof, Here, the antibody, antibody fragment, or a combination thereof binds to the cell surface of endothelial progenitor cells or endothelial cells. In one embodiment, the cell surface antigen is CD133, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, VEGFR -3, Muc-18 (CD146), Thy-1, Thy-2, CD130, CD30, Stem cell antigen (Sca-1), stem cell factor 1 (SCF / c-Kit ligand), Tie-1 , Tie-2, VE-cadherin, P1H12, TEK, CD31, Ang-1, Ang -2, HAD-DR, CD45, CD14, CD105, E-selectin, von Bilbrom vWF, or a combination thereof.

[0039] The medical device includes a blood-contacting surface (or luminal surface) for application of the coating. The ligand (e.g., antibody and / or antibody fragment) can bind to endothelial progenitor cells (EPCs), etc. interact with antigens on target cells, immobilizing endothelial progenitor cells on the surface of the device and forming endothelium. possible.

[0040] Ligands bind to cell membrane structures such as receptor molecules on circulating endothelial cells and / or endothelial progenitor cells. For example, the ligand may be an antibody, an antibody fragment, a peptide, or the like. The target molecule may be a small molecule such as a phosphodiesterase inhibitor, a cell adhesion molecule, a basement membrane component, or a combination thereof. In embodiments using antibodies, the antibody binds to a specific target, such as a cell surface receptor on the cell membrane of the cell. Ligands also recognize and bind to epitopes or structures such as fatty acids, peptides, and proteins. They may be derived from a variety of sources, such as cellular components including proteins, nucleic acids, saccharides, etc. For example, antibodies can interact with structures such as antigens on the surface of progenitor endothelial cells. To produce a result or effect.

[0041] The ability of antibodies to bind to target proteins at solid-liquid interfaces is a key factor in antibody-based in vitro diagnostic assays. The Fab domain of the immobilized antibody binds to the antigen, which is of primary importance for assays and in vivo therapeutics. To be compatible, the Fab domain must (i) be accessible, i.e., (ii) are biologically active, i.e., bind to target molecules; The activity of immobilized antibodies varies depending on the molecular structure and the dissociation constant (Kd) of the immobilized antibody. Antibodies with more accessible Fab domains are sensitive to variations in their chemical properties. , showing higher activity than randomly immobilized antibodies. Atomic force microscopy, neutron reflectometry, Several techniques, including but not limited to spectroscopic ellipsometry, and mass spectrometry can be used to determine the activity, accessibility, and orientation of immobilized antibodies . Saha et al.Analyst,2017,142,4247-4256. fixed A quantitative radiolabeling assay was also used to determine the accessibility of the Fab domain. It is possible. Ibid.

[0042] The ligand / biomolecule ensures the accessibility of the active site of the ligand / biomolecule This allows the ligand / biomolecule to be immobilized on the medical device in an oriented manner. Binding of ligands / biomolecules at specific sites separate from / different from the active site In one embodiment, the ligand / biomolecule (e.g., an antibody or fragments) may contain their active sites (e.g., Fab regions or domains, antigen-binding sites or The antibody may be immobilized on the medical device via a site on the outside of the antibody domain.

[0043] For example, the ligand / biomolecule (e.g., antibody or antibody fragment) of the coating may be At least 100% of the total active sites of the targeting ligand / biomolecule (e.g., antibody or antibody fragment) at least 1%, at least 3%, at least 5%, at least 8%, at least 10%, At least 15%, at least 20%, at least 25%, at least 30%, at least 35 %, at least 40%, at least 45%, at least 50%, at least 60%, at least At least 70%, at least 80%, or at least 90% of their active sites (e.g., , Fab region or domain, antigen binding site or domain) For example, Fab accessibility.

[0044] In one embodiment, the ligand / biomolecule (e.g., antibody or antibody fragment) of the coating is applied to a coating (e.g., a polydopamine coating) in an unoriented manner. the active sites (e.g., F) of the attached ligands / biomolecules (e.g., antibodies and antibody fragments) ab region or domain, antigen-binding site or domain) is about 5 times more accessible %, approx. 8%, approx. 10%, approx. 15%, approx. 20%, approx. 25%, approx. 30%, approx. 35%, approx. 40% , about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, at least 1%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15% , at least 20%, at least 25%, at least 30%, at least 35%, less At least 40%, at least 45%, at least 50%, at least 60%, at least 70% %, at least 80%, or at least 90% larger than their active sites (e.g., Fa b region or domain, antigen binding site or domain) accessibility.

[0045] In other embodiments, at least 1%, at least 3%, at least 5%, at least 8%, %, at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 5 0%, at least 60%, at least 70%, at least 80%, or at least 90 % of the ligands / biomolecules have accessible active sites (e.g., Fab regions or In other words, the ligand / biome At least 1%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, At least 35%, at least 40%, at least 45%, at least 50%, at least At least 60%, at least 70%, at least 80%, or at least 90% of the active sites ( For example, a Fab region or domain, an antigen binding site or domain) is blocked. It is not modified or denatured. This is due to the specific site away from the active site of the ligand / biomolecule. This can be achieved by binding the ligand / biomolecule with

[0046] In yet other embodiments, at least 1%, at least 3%, at least 5%, or at least At least 8%, at least 10%, at least 15%, at least 20%, at least 25% , at least 30%, at least 35%, at least 40%, at least 45%, less at least 50%, at least 60%, at least 70%, at least 80%, or at least As much as 90% of antibodies and / or antibody fragments are available to bind to cell surface antigens.

[0047] The accessibility of the Fab and / or Fc domains of the immobilized antibodies is discussed in Saha et al. Analyzed according to et al. Analyst 142:4247-4256 (2017) Fab domain accessibility assay - a known amount of antibody is coated on the The device (e.g., disk, ePTFE graft, stent) is attached to the conjugated antibody. Alternatively, the antibody may be incubated with a molar excess (relative to the molar amount of bound antibody) of the antigen that can bind to the antibody. Using a molar excess will result in saturation of the available antibody domains. After application, the coated device is washed and then incubated with antibodies that bind to epitopes different from the primary antibody. A secondary radiolabeled (e.g., 125I-labeled) antibody is added in a molar excess (relative to the amount of bound antibody). A stock of different known concentrations of radiolabeled secondary antibody in solution may be added in excess. The bound secondary radioactive material in the Fab accessibility assay can then be used as a reference. The amount of labeled antibody was determined by subtracting the final signal after binding of the secondary radiolabeled monoclonal antibody from the total antibody content. It may be calculated by subtracting the signal of the coated device. et al.Analyst 142:4247-4256(2017). of immobilized antibodies Other techniques used to determine activity, accessibility, and orientation are atomic forces Includes microscopy, neutron reflectometry, spectroscopic ellipsometry, and mass spectrometry.

[0048] Cell adhesion can be assessed using a suitable method, such as a cell adhesion assay. Adherent cells can be quantified using colorimetric or fluorescent detection.

[0049] The present disclosure provides a prosthetic heart valve or prosthetic venous valve having a coating comprising polydopamine. In some embodiments, the valve is a prosthetic aortic valve, a prosthetic pulmonary valve, a prosthetic mitral valve, a prosthetic tricuspid valve, or a prosthetic thoracic valve. It is a cusp valve.

[0050] The antibodies and / or antibody fragments can be monoclonal or polyclonal. In one embodiment, the antibody and / or antibody fragment is a Fab or F(ab')2 fragment. The antibodies and / or antibody fragments may specifically bind to different cell surface antigens.

[0051] The linker can be hetero- or homobifunctional. After ligation, a linker molecule may be used to covalently link one or more types of antibodies. The linker provides several functionally active groups to the matrix. The esterification can be achieved by direct (i.e., through the catechol group) or by esterification, amidation, or acylation. The linker molecule can be attached through known coupling chemistries such as amine-carbonylation. The polydopamine coating is attached through the direct formation of saturated and unsaturated bonds. and amines that are available for reaction with a ligand (e.g., an antibody and / or antibody fragment). The functional groups provided can be di-, tri-, or tetra-amine functional compounds. For example, the linker molecule may be polyethylene glycol (PEG), polyethyleneimine (PEI), ), polyallylamine (PALLA), or PEG derivatives (e.g., mPEG-succinic acid). poly(ethylene glycol) copolymers such as PEG-N-hydroxysuccinimide or mPEG-N-hydroxysuccinimide The polymer may be an amine-functional polymer. ner et al.,Influence of a poly-ethyleneg lycol spacer on antigen capture by immob ilized antibodies.J.Biochem.Biophys.Meth See, J.D.Ods 45:211-219 (2000). Mixtures of polymers may also be used. These molecules may be fused to one or more ligands (e.g., antibodies and / or antibody fragments). It contains multiple pendant amine functional groups that can be used to surface immobilize fragments (e.g., fragments).

[0052] Coatings on medical devices may contain pharmaceutical substances that inhibit smooth muscle cell migration and / or proliferation. In certain embodiments, the pharmaceutical agent may further comprise any of the following: paclitaxel, rapamycin, thiazolinone ... mycin, rapamycin derivatives, sirolimus, everolimus, tacrolimus, biolimus The medicinal substance is a vasodilator. It is possible.

[0053] Coated medical devices may be used for targeted local drug delivery (e.g., of pharmaceutical substances) and / or or systemic therapy.

[0054] A medical device is a device that is temporarily or permanently administered to a mammal for the prevention or treatment of a medical condition. These devices can be subcutaneous, percutaneous, or surgically introduced. and introduced into an organ, tissue, or organ such as an artery, vein, or the ventricles and / or atria of the heart. Medical devices include stents, stent grafts, and any other medical device placed within a lumen of a liver. Polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE) ), or covered stents, artificial heart valves, such as those covered with artificial vascular grafts; Fixtures for connecting artificial hearts and prosthetic organs to vascular circulation, venous valves, abdominal aortic aneurysms (A AA) Grafts, inferior vena cava filters, permanent drug infusion catheters, embolization coils, vascular embolization Embolic materials used in the procedure (e.g., cross-linked PVA hydrogel), vascular sutures, and vascular anastomotic fixation devices, transmyocardial revascularization stents and / or other conduits.

[0055] The present disclosure provides a prosthetic heart valve or prosthetic venous valve having a coating comprising polydopamine. In some embodiments, the valve is a prosthetic aortic valve, a prosthetic pulmonary valve, a prosthetic mitral valve, or It is an artificial tricuspid valve.

[0056] The coating of the prosthetic valve may contain pharmaceutical substances that inhibit smooth muscle cell migration and / or proliferation. In certain embodiments, the pharmaceutical agent may further comprise a pharmaceutical agent selected from the group consisting of paclitaxel, rapamycin, and cefotaxime. Isin, rapamycin derivatives, sirolimus, everolimus, tacrolimus, biolimus , Biolimus A-9, or a combination thereof.

[0057] In another embodiment, the method includes implanting the medical device in a patient in need thereof. Methods are provided for treating vascular diseases, including restenosis and atherosclerosis. The method includes implanting a medical device having the coating in a blood vessel or hollow organ of a patient. This includes:

[0058] The term "endothelial progenitor cells" refers to cells that have the potential to differentiate into mature, functional endothelial cells. These include cells of any lineage. For example, endothelial progenitor cells can be derived from bone marrow, blood, or local tissue sources. in endothelial cells at any developmental stage, from progenitor or stem cells to mature functional endothelial cells. Endothelial progenitor cells are genetically modified, non-malignant cells. Endothelial colony-forming cells (EPCs) Endothelial colony forming cells may include CFCs and myeloid angiogenic cells (MACs). D31+, CD105+, CD146+, CD45-, and / or CD145- Endothelial colony forming cells have the inherent ability to form tubes in vitro and in vivo. Endothelial colony forming cells are the building blocks for new blood vessel formation or vascular repair. Myeloid angiogenic cells can be CD45+, CD14+, CD31+, C The medium can be D146- and / or CD34-. MAC-derived parasites can enhance endothelial network formation in vitro and in vivo. Clin factors can be stimulators of angiogenesis. Medina et al., Endothelial Progenitors:A Consensus Stat ement on Nomenclature,Stem Cells Transla tional Medicine,2017;6:1316-1320.

[0059] Fully differentiated internal use for in vitro studies or use of coated medical devices Endothelial cells can be isolated from arteries or veins, such as the human umbilical vein, while endothelial progenitor cells can be isolated from peripheral arteries or veins. Endothelial cells can be isolated from blood or bone marrow. Endothelial cells can be isolated from medical devices having a coating of the present invention. In another embodiment, the endothelial cells are attached to the medical device by incubation with the endothelial cells. The endothelial cells may be transformed / transfected endothelial cells.

[0060] The ligand may be used in place of or in combination with an antibody or antibody fragment. The compound may be a small molecule, including a synthetic or naturally occurring molecule or peptide, that can be used in combination with other compounds. For example, lectins are naturally occurring carbohydrate-binding peptides of non-immune origin. cell-specific lectin antigen (Ulex Europaeus Uea 1) (Schatz et al.2000 Human Endometrial Endothelial Cells:Isolation,Characterization,and In Flammatory-Mediated Expression of Tissue Factor and Type 1 Plasminogen Activator Inhibitor. Biol Reprod 62:691-697) is a precursor endothelial It can selectively bind to the cell surface of cells. Synthetic small molecules bind to various cell surface receptors. These molecules are created to target specific receptors. Selectively bind and target specific cell types, such as endothelial progenitor cells and / or endothelial cells. Small molecules can be synthesized to recognize endothelial cell surface markers such as VEGF. For example, SU11248 (Sugen) (Mendel et al. 2014) 03 In vivo antitumor activity of SU11248 ,a novel tyrosine kinase inhibitor target ting vascular endothelial growth factor and platelet-derived growth factor recep tors:determination of a pharmacokinetic / pharmacodynamic relationship.Clin Cancer Res.January;9(1):327-37), PTK787 / ZK22258 4(Drevs J.et al.2003 Receptor tyrosine k inases:the main targets for new anticanc er therapy.Curr.Drug Targets.February;4( 2):113-21), and SU6668 (Laird, AD et al. 200 2 SU6668 inhibits Flk-1 / KDR and PDGFRbet a in vivo,resulting in rapid apoptosis o f tumor vasculature and tumor regression in mice, FASEB J. May;16(7):681-90) In another embodiment, a synthetic small molecule that targets the endothelial cell surface is used. Another subset of children are those who have been treated with, for example, alpha(v)beta(3) integrin inhibitors, S5 M256, and SD983 (Kerr J S. et al. 1999 Nov el small molecule alpha v integrin antag onists:comparative anti-cancer efficacy with known angiogenesis inhibitors can b e used.{j}Anticancer Res March-April;19( 2A)-959-68). Both SM256 and SD983 bind to the surface of endothelial cells. It is a synthetic molecule that targets and binds to the alpha(v)beta(3) present.

[0061] In one embodiment, the substrate / medical device (cobalt chrome, stainless steel, ePTFE, and and / or polystyrene, etc.) is a polydopamine membrane The amine-functionalized polyethylene glycol is coated with polydopamine. Deposited on the coated substrate. Functionalized ligands / biomolecules are introduced to form functional It reacts with PEG.

[0062] In another embodiment, the ligand / biomolecule (e.g., antibody or antibody fragment) is a polydote. For example, freshly prepared polydopamine is directly immobilized on the coating. The coated substrate / medical device is then incubated with the unmodified antibody (or antibody fragment) in a buffer solution (e.g., PBS). The antibody (or antibody fragment) coated substrate / medical device is then exposed to the solution. Rinse thoroughly with buffer (e.g., PBS) to remove adsorbed antibody.

[0063] The coated medical device captures / binds native / normal cells or genetically modified cells. Genetically modified cells can be constitutively engineered as described herein to Or, when stimulated to do so, may secrete a pharmaceutical substance.

[0064] In one embodiment, circulating endothelial progenitor cells are captured and distributed on the lumen or blood-contacting surface of the device. Immobilized and capable of restoring, enhancing or accelerating the formation of functional endothelium at the device implantation site The target cell can be a cell.

[0065] In another embodiment, the ligand / biomolecule (e.g., antibody or antibody fragment) is attached to the cell surface. Cell surface antigens / molecules of genetically engineered cells that have been genetically engineered to express the antigen / molecule By recognizing only the molecule, genetically modified cells (e.g., mammalian cells such as human cells) Binding of target cells to the ligand / biomolecule binds the cells to the device. It may be immobilized on a surface.

[0066] In this way, only the genetically modified cells can bind to the surface of the medical device.

[0067] The ligand / biomolecule (e.g., antibody or antibody fragment) can bind to CD133, CD34, CD 14, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Mu c-18 (CD146), CD130 stem cell antigen (Sca-1), stem cell factor 1 (SCF / c-Kit ligand), cell surface antigens such as Tie-2, H-2K k and HLA-D It can be specific for binding to MHC, such as R, or synthetic antigens.

[0068] In one embodiment, EPCs are engineered to express vasodilators, e.g., to enhance the flow of epicardial coronary arteries. It has been genetically modified to promote dose-dependent positive remodeling.

[0069] Melanin, melanin-like polymers, synthetic versions of melanin, or aromatic catechols Polymers include polydopamine, polymers of dopamine analogues, eumelanin, and pheomelanin. Melanin and neuromelanin are examples of melanin-specific proteins.

[0070] Polydopamine Polydopamine is formed by polymerization of monomeric dopamine. Polydopamine (PDA) is a polymer formed by the oxidative self-polymerization of dopamine under slightly basic conditions. In one embodiment, the PDA membrane is a synthetic eumelanin polymer formed via The dopamine-containing material may be formed by immersing the material / medical device in an aqueous solution of dopamine.

[0071] The exact structure of polydopamine is not well understood, and several structures have been proposed. There are.

[0072] Polymerization of dopamine can occur under oxidative conditions. Exposure to air (i.e., oxygen) In one embodiment, the initial oxidation of dopamine is sufficient to initiate polymerization. This occurs at the ion moiety and then reacts with another molecule of dopamine or (pendant The nitrogen-containing bicycle can be formed via intermolecular cyclization (via a primary amine). One structure (Structure A) (described in WO 2010 / 006196) is a polydopamine The amine is a repeating 5,6-dihydroxy-3H-indole bridged through positions 4 and 7. Another structure (structure B, Zhao et al., Pol Chem., 2010, 1, 1430-1433) shows a similar polymer. It is suggested that every other 5,6-dihydroxy-3H-indole unit is 5,6-dihydroxy-3H-indole. Structure C is another example of a polydopamine-related A possible structure has been proposed, which is also similar to structure A, but contains a 5,6-dihydro Every other hydroxy-3H-indole unit is replaced by a non-cyclized dopamine molecule (U.S. Patent No. 9,272,075). Therefore, this structure of polydopamine is a primary Contains amine functionality. Structure D (Kang et al., Langmuir, 2009, 2 5, 9656-9659) has also been proposed, which shows that the five-membered nitrogen ring This structure suggests that the quinone ring is attached to the catechol ring as well as the catechol ring. Finally, structure E (Dreyer et al. al. Langmuir, 2012, 28, 6428-6435) is a polydopa The amine is not a covalently bonded polymer, but instead is primarily composed of 5,6-dihydroxyindoline. and its dione derivatives, which are supramolecular assemblies of monomers with completely different structures. is doing.

[0073] In the context of this disclosure, representations of the structure of polydopamine are used in the methods and coatings of the present invention. The above discussion is included merely for background reference and is not essential to the implementation of the present invention. Please note.

[0074] As referred to herein, "polydopamine" refers to a compound containing dopamine and / or dopamine. In one embodiment, polydopamine is suitably formed by polymerization of dopamine analogs. Dopamine analogs are compounds that are the same as or similar to dopamine. Molecules involved in related biochemical pathways, and structures of dopamine, including oxidized derivatives of tyrosine In one embodiment, the dopamine analog is a compound of formula (I): , where R 1 ~R 9 One or more of the following is not H: [ka]

[0075] In another embodiment, the dopamine analog is a compound of formula (I), wherein R 1 ~R 9 is H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -OH, -CO2H, -C(O)C1-C8 alkyl, -C(O)C2-C8 alkenyl, -C( O) C2-C8 alkynyl.

[0076] Naturally occurring dopamine analogs include: [ka]

[0077] Other exemplary dopamine analogs are illustrated below: [ka]

[0078] Method for preparing polydopamine coatings Dopamine in alkaline aqueous solution exposed to air (i.e., oxygen) reacts with additional reactants However, the polymerization rate is slower when the solution is dissolved in water. The oxidation current can be enhanced by the addition of chemical oxidants or by the addition of dopamine. Oxidizing agents include, but are not limited to, ammonium persulfate and sodium persulfate. Thus, in one embodiment, the polydopamine surface coating can be used to protect the surface of the substrate from acid. contacting the compound with a mixture comprising a compounding agent and dopamine and / or a dopamine analog; is formed by

[0079] Polymerization of dopamine also likely involves deprotonation and acidification of the catechol hydroxyl groups. It has been observed that the activation of oxidizing agents is faster in alkaline aqueous solutions. The use of dopamine is based on the idea that polymerization of dopamine occurs within a reasonable time frame at neutral pH or even acidic pH. H. A suitable oxidizing agent is ammonium persulfate. and sodium persulfate. U.S. Patent No. 9,272,075.

[0080] In one embodiment, the polydopamine surface coating is at a pH between 4 and 10, e.g., pH >7 or pH 7, the surface of the substrate is treated with an oxidizing agent and dopamine and / or In another embodiment, the polydode is formed by contacting the polydode with a mixture containing the polydode. The surface coating of methylparaben is formed at a pH of <7, e.g., pH 4-7. In an embodiment, the polydopamine surface coating has a pH of 5 to 6.9, for example, pH 5. The pH of the dopamine and / or dopamine analog solution is 5 to 6.5. These can be adjusted using any suitable acid or base, such as HCl or NaOH, respectively. The pH of the solution can be adjusted by adding a suitable buffer, e.g., MES, ACES, PIPES, MO PSO, Bis-Tris Propane, BES, MOPS, TES, and HEPES buffers can be controlled using

[0081] The amount of oxidizing agent affects the rate of polymerization. In one embodiment, the amount of dopamine in the solution is 1 g / L to 5 g / L, and the amount of ammonium persulfate (APS) in the solution is 0.6 g / L In another embodiment, 1 g / L dopamine and 0.6 g / L AP S is used for polymerization. The polymerization rate increases with increasing dopamine and / or APS concentrations. In some embodiments, the concentration of dopamine or an analog may be increased by 0.5 to 100 mg / kg. The concentration of APS can be 0.1 to 5 g / L.

[0082] Dopamine polymerization can be carried out in aqueous solution or in methanol, ethanol, propanol, and and / or in an aqueous / organic mixture, such as a mixture of isopropanol and water.

[0083] The time required to form the polydopamine coating will vary depending on the specific reaction conditions used. For example, the addition of an oxidizing agent can accelerate polymerization or cause the polymerization to become neutral or Polydopamine coatings may even allow for the use of acidic pH. For example, desirable polydopamine-containing The time is 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes , can be formed within 10, 5, or 2 minutes. .,Langmuir 2013,29(27),8619-8628. As a general principle The longer the polymerization time, the thicker the polydopamine coating that is formed. Therefore, the optimal time for polymerization of dopamine is long enough to obtain sufficient coverage of polydopamine. but for a long enough time to allow uncontrolled polydopamine particles to form in solution. In some embodiments, the polymerization time is 24 hours or less, for example, up to 12 hours, for example, up to 6 hours. , 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 10 minutes, 5 minutes, or 2 minutes In one embodiment, post-processing techniques such as sonication are used to separate polydopamine aggregates and and particles can be removed.

[0084] Polydopamine coatings can be formed at room temperature, but polymerization can be slower or faster. It can be done at temperature.

[0085] The thickness of the polydopamine coating is about 0.1 nm to about 10 nm, about 1 nm to about 50 nm, about 1 nm to about 40 nm, about 1 nm to about 30 nm, about 1 nm to about 20 nm, about 1 nm ~about 15nm, about 1nm to about 10nm, about 1nm to about 100nm, about 5nm to about 80nm , about 6nm to about 60nm, about 10nm to about 50nm, about 10nm to 30nm, about 0.1μ The diameter may range from about 1 μm to about 150 μm, or from about 1 μm to about 100 μm. r et al., Langmuir 2013, 29(27), 8619-8628.

[0086] A possible alternative approach to forming polydopamine using charge (voltage) is proposed by Kang et al. et al.Angewandte Chemie,2012,vol.124,pp It is described in 1-5.

[0087] Prior to coating, the surface of the substrate was cleaned to improve adhesion to the polydopamine. Pre-cleaning or pre-treating the surface can also improve coating uniformity. can be improved.

[0088] Suitable cleaning or pre-treating agents include ethanol or isopropanol (IPA), Solvents include mixtures of alcohols and aqueous solutions of hydroxide compounds (e.g., sodium hydroxide) High pH solutions such as sodium hydroxide solution itself, tetramethylammonium hydroxide Solutions containing tetrahydrofuran (TMAH), basic piranha (ammonia and hydrogen peroxide), acidic piranha Rania (a mixture of sulfuric acid and hydrogen peroxide), and sulfuric acid and potassium permanganate Other oxidizing agents, including peroxosulfuric acid or peroxodisulfuric acid solutions of different types (e.g., ammonium, sodium, and potassium salts, e.g., also as ammonium persulfate), or a combination thereof.

[0089] Two specific pretreatment methods are described - Method A and Method B. Method A involves pretreating the substrate with isopropanol. In Method B, the substrate is treated with isopropanol and then In one embodiment, the polydopa is treated with a solution of APS (ammonium persulfate). Prior to forming the surface coating of the metal, the surface of the substrate is pretreated with an oxidizing agent. In an embodiment, the surface of the substrate is treated with isoprene prior to forming the polydopamine surface coating. In a further embodiment, the surface of the polydopamine is treated with propyl alcohol and an oxidizing agent. Before forming the coating, the surface to be coated is treated with isopropanol and persulfate. Pretreated with ammonium.

[0090] The polydopamine layer may contain, for example, alkene and / or alkyne groups or thiol groups. Such polydopamine surfaces may be functionalized with at least a proportion of alkene and / or alkyne or thiol group functionalized dopamine (or analogues) Synthetic dopaminergic compounds can be prepared by polymerization of dopamine and dopamine analogs, including: Analogs can be formed by functionalizing the primary amine of dopamine.

[0091] After polydopamine film formation, the substrate / medical device is coated with biomolecules (e.g., proteins). Further functionalization with molecules containing amine and / or thiol groups, which are common moieties Biomolecules can be purified under very mild conditions (e.g., near-neutral pH or neutral pH). can be immobilized at pH 6.0 and room temperature.

[0092] Organic Polymers / Oligomers The polydopamine and the ligand (e.g., antibody and / or antibody fragment) may be prepared by the addition of an organic polymer. They may be linked via a linker such as a mer / oligomer.

[0093] Non-limiting examples of organic polymers include polyether derivatives (e.g., polyethylene glycol). Polyethylene glycol (PEG), polyethylene glycol (PEG) derivatives, polypropylene glycol ( PPG or polypropylene glycol (PPG) derivatives), polysilicon, polydimethylsilane Siloxane, siloxane derivatives, polyurethane, protein, peptide, polypeptide Hyaluronic acid, hyaluronic acid derivatives, poly-N-vinylpyrrolidone, poly-N-vinyl pyrrolidone derivatives, polyethylene oxide, polyethylene oxide derivatives, polyalkoxy Polyethylene glycol, polyglycidol, polyvinyl alcohol, polyvinyl alcohol derivatives Body, polyacrylic acid, polyacrylic acid derivatives, silicone, silicone derivatives, polysaccharide Carides, polysaccharide derivatives, polysulfobetaines, polysulfobetaine derivatives, Polycarboxybetaine, polycarboxybetaine derivatives, polyHEMA, etc. Alcohol, polyacids such as alginic acid, dextran, agarose, polylysine, polymer Acrylic acid, polymethacrylic acid derivatives, polymethacrylamide, polymethacrylamide derivatives Conductors, polyacrylamide, polyacrylamide derivatives, polysulfone, polysulfone derivatives Conductors, sulfonated polystyrene, sulfonated polystyrene derivatives, polyallylamine, poly Triarylamine derivatives, polyethyleneimine, polyethyleneimine derivatives, polyoxazo polyamines, polyamine derivatives, and combinations thereof. Block copolymers of the above polymers are also useful, for example, poly(vinyl alcohol) -co-ethylene), poly(ethylene glycol-co-propylene glycol), poly (vinyl acetate-co-vinyl alcohol), poly(tetrafluoroethylene-co -vinyl alcohol), poly(acrylonitrile-co-acrylamide), poly(acrylonitrile-co-acrylamide), Examples include acrylonitrile-co-acrylic acid-co-acrylamidine.

[0094] In certain embodiments, the organic polymer is hyaluronic acid, a hyaluronic acid derivative, poly-N- Vinylpyrrolidone, poly-N-vinylpyrrolidone derivatives, polyether derivatives (e.g., Polyethylene glycol (PEG), polyethylene glycol (PEG) derivatives, poly Propylene glycol (PPG) or polypropylene glycol (PPG) derivatives, The polymer may be a polyvinyl alcohol, a polyvinyl alcohol derivative, or a combination thereof. In embodiments, the organic polymer is polyethylene glycol (PEG), polyethylene glycol PEG derivatives, polypropylene glycol (PPG), polypropylene glycol (PPG) derivatives), or combinations thereof. - (e.g., copolymers of ethylene glycol and propylene glycol), their terpolymers Polymers and mixtures thereof are also contemplated.

[0095] Organic polymers that may be used in the present disclosure include PEG, polylactate, polylactic acid, sugars, lipids , polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), and combinations thereof. Attachment of organic polymers to the medical device or medical device can be achieved by ionic, hydrogen, hydrophobic, coordination, This can be achieved by covalent or non-covalent bonding, such as adhesion and physical absorption.

[0096] Polyether polymers contain hydroxyl groups, or amino and thiol groups. It may be terminated with other end groups, including but not limited to these.

[0097] The linker (e.g., organic polymer) may be any suitable linkage / bond ( The polydopamine may be linked to the polydopamine via a bond. Under very mild conditions (e.g., neutral pH and / or room temperature), Michael addition or Schiff addition occurs. It can be functionalized with thiol- or primary amine-containing molecules via base formation.

[0098] Heterobifunctional organic polymer (e.g., PEG) chains are amine and thiol functional groups in combination with cysteine, thiazolinone, and / or biotin. In one embodiment, PEG can be attached to the medical device by physical adsorption or covalent bonding. Can be grafted onto a surface 81 In another embodiment, the amine-PEG-alkyne is is immobilized on the coated medical device, and a ligand containing an azide functional group (e.g., In yet another embodiment, polydopamine is , a thiolated linker (e.g., a thiolated organic polymer such as a thiolated PEG), linked to an aminated linker (e.g., an aminated organic polymer such as aminated PEG) PEG may be used in conjunction with melanin, melanin-like polymers, synthetic versions of melanin, or or aromatic catechol polymers (e.g., polydopamine or poly(dopaminergic) analogs) binding to a ligand / biomolecule (e.g., antibody and / or antibody fragment) Other functional groups for forming bonds include maleimides and alkenes.

[0099] Organic polymers (e.g., polyether derivatives such as PEG) can be attached to polydopamine. for attachment to ligands (e.g., antibodies and / or antibody fragments) The medical device may have multiple functional groups. Various types of functionalized organic polymers (e.g., The organic polymer may be covalently or non-covalently bonded. It can be covalently attached to polydopamine.

[0100] In one embodiment, functionalized PEG amines (hydrazides or dibenzocyclooctynes (D BCO (functionalized) or aminated PEG can be used to conjugate polydopamine. The dibenzocyclooctyne surface was coated with a solution of amino-PEG-DBCO on a substrate or medical device. By immersing the device in the PDA, a PDA is formed on the coated substrate or medical device. do.

[0101] In one embodiment, the organic polymer is difunctionalized with amine and / or sulfhydryl groups. will be done.

[0102] Polyethylene glycol (PEG) PEG is a polyether compound which, in its linear form, has the general formula H[O—CH2— CH2] n Branched PEGs, including hyperbranched PEGs and dendritic PEGs, are also contemplated. and are generally known in the art. Typically, branched polymers have a central branch core moiety. and multiple linear polymer chains attached to a central branch core. PEGs are generally Glycerol, glycerol oligomers, pentaerythritol, and sorbitol, etc. It is used in branched forms that can be prepared by the addition of ethylene oxide to various polyols. The central branch can also be derived from several amino acids, such as lysine. PEG-OH) is a PEG-OH copolymer with the general form R(-PEG-OH). m where R is , derived from core moieties such as glycerol, glycerol oligomers, and pentaerythritol and m represents the number of arms. 3,575, U.S. Patent No. 5,229,490, U.S. Patent No. 4,289,872, U.S. Patent No. Patent Application Publication No. 2003 / 0143596, International Publication No. 96 / 21469, and International Multi-arm PEG molecules such as those described in Publication No. 93 / 21259 may also be used. It is possible.

[0103] PEG is from about 100 daltons to about 20,000 daltons, from about 200 daltons to about 10, 000 Daltons, about 200 Daltons to about 5,000 Daltons, about 250 Daltons to about 8, 000 Daltons, about 200 Daltons to about 6,000 Daltons, about 300 Daltons to about 5, 000 Daltons, about 200 Daltons to about 400 Daltons, about 200 Daltons to about 300 Daltons daltons, or in the range of about 500 daltons to about 1,000 daltons. .

[0104] The coating may contain two or more PEG molecules with different average molecular weights.

[0105] When immobilized on a substrate, PEG effectively prevents nonspecific binding of proteins to the substrate. Possible.

[0106] PEG may contain amine and / or thiol groups that may be reactive to polydopamine coatings. In addition, PEG chains can be functionalized with hydrazide, azide, cyclooctyl, and methyl groups. The PEG may be further modified to include PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, Examples of functionalized PEGs are shown below. [ka] [ka] [ka]

[0107] In combination with polydopamine functionalization, PEG can be applied by simple dip coating under mild conditions. The deposition can be carried out via a film.

[0108] PEG has been shown to remain stable in the body for extended periods of time with minimal degradation. Stability limits inflammation due to particulate formation and contributes to the overall biocompatibility of the material.

[0109] antibody The ligand (e.g., antibody and / or antibody fragment) may be attached via any suitable linkage. ge) / bond through a linker (e.g., organic polymer) or polydopa In one embodiment, the ligand (e.g., an antibody and / or an anti- The fragments are attached to a linker (e.g., an organic polymer) or polydopamine. It has exposed sugars so that they can be oxidized.

[0110] The coating of the medical device may further comprise an antibody, an antibody fragment, or a combination thereof. The antibody, antibody fragment, or combination thereof binds to cell surface antibodies of endothelial progenitor cells or endothelial cells. In one embodiment, the cell surface antigen is CD133, CD34, CD45, CD31, CD14, CDw90, CD117, HLA-DR, VEGFR- 1, VEGFR-2, VEGFR-3, Muc-18(CD146), Thy-1, Th y-2, CD130, CD30, stem cell antigen (Sca-1), stem cell factor 1 (SCF / c -Kit ligand), Tie-1, Tie-2, VE-cadherin, P1H12, TEK , CD31, Ang-1, Ang-2, HAD-DR, CD45, CD14, CD105 , E-selectin, or a combination thereof. The cell surface antigen is H-2K k and H It can be an MHC such as LA-DR.

[0111] In one embodiment, an antibody and / or a peptide that specifically binds to CD34 and / or CD133 is or antibody fragments are used. The hybridomas were collected from the American Type Tissue Collection In another embodiment, VEGFR- 1 and antibodies that specifically bind to VEGFR-2, CD133, or Tie-2, and and / or antibody fragments are used.

[0112] The antibody, antibody fragment, or combination thereof can be monoclonal. , antibody fragments, or combinations thereof can be polyclonal. The antigen-binding portion may be a humanized antibody, a human antibody, a monoclonal antibody, a chimeric antibody, a polyclonal antibody, or a antibodies, recombinantly expressed antibodies, and antigen-binding portions of the foregoing. Not limited to these.

[0113] The antigen-binding portion of the antibody specifically binds to a cell surface antigen of an endothelial progenitor cell or an endothelial cell. It may comprise a portion of an antibody that

[0114] The antibody, antibody fragment, or combination thereof may include Fab or F(ab')2 fragments. Antibodies, antibody fragments, or These combinations may specifically bind to the same cell surface antigen or may bind to different cell surface antigens. In certain embodiments, the antibody, antibody fragment, or combination thereof is capable of binding to a medical device. When the device is implanted in a subject, it captures endothelial progenitor cells and / or endothelial cells in vivo. do In certain embodiments, the antibody, antibody fragment, or combination thereof is an organic molecule described herein. It contains exposed sugars that can be oxidized for attachment to an intermediate linker, such as a polymer.

[0115] Antibodies or antigen-binding portions thereof in which specific amino acids have been substituted, deleted, or added are also disclosed herein. These alterations have substantial effects on the biological properties of the peptide, such as binding activity. It has no significant effect.

[0116] The peptide may be, for example, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or less than about 1% of amino acid residues are substituted or deleted, but bind to cell surface antigens and the like, which retain essentially the same immunological properties, including but not limited to: The disclosed antibodies can be functionally active variants of their antigen-binding portions.

[0117] An antibody or antigen-binding portion thereof has biological activity, e.g., binding of an antigen, such as a cell surface antigen. It may also include variants, analogs, orthologs, homologs, and derivatives of peptides that exhibit binding. Peptides may contain one or more analogs of an amino acid (e.g., a non-naturally occurring amino acid, an unrelated amino acid, or an unrelated amino acid). Amino acids that occur naturally only in biological systems, modified amino acids from mammalian systems, peptides with substituted bonds, and peptides with substituted bonds known in the art. It may contain other modifications as well.

[0118] An antibody or antigen-binding portion thereof may be derivatized or conjugated to another functional molecule. For example, the antibody may be linked to another antibody, a detectable agent, an immunosuppressant, a cytotoxic agent, a pharmaceutical product, binding to another molecule (such as a streptavidin core region or a polyhistidine tag) Intermediate proteins or peptides, amino acid linkers, signal sequences, immunogenic carriers or glutathione-S-transferase, histidine tag, staphylococcal protein to one or more other molecular entities, such as ligands useful for protein purification, such as cyclopentasiloxane A (chemical The nucleic acid may be operably linked (by means of electrical coupling, genetic fusion, non-covalent interactions). Cytotoxic agents include radioisotopes, chemotherapeutic agents, and drugs of bacterial, fungal, plant, or animal origin. Such cytotoxic agents may include toxins, such as enzymatically active toxins, and fragments thereof. can be coupled to the antibodies of the present disclosure using standard procedures, e.g., for use in antibody-based therapy. The compounds may be used to treat patients in need thereof.

[0119] One type of derivatized protein is two or more proteins (of the same or different types). Suitable cross-linkers are prepared by cross-linking the Those that are heterobifunctional, with two separate reactive groups attached (e.g., m-maleimide amides) benzoyl-N-hydroxysuccinimide ester), or homobifunctional ( Derivatizing (or labeling) proteins includes, for example, disuccinimidyl suberate. Useful detectable agents include fluorescers, various enzymes, prosthetic groups, luminescent materials, and bioluminescence. Non-limiting exemplary fluorescent detectable agents include fluorescein, Contains fluorescein, fluorescein isothiocyanate, rhodamine, and phycoerythrin .

[0120] The antibody or antibody fragment may be a monoclonal antibody, a polyclonal antibody, a humanized antibody, or or chimeric antibodies, or combinations thereof.

[0121] The antibody or antibody fragment is intended to inhibit the attachment of circulating endothelial progenitor cells and / or endothelial cells to a medical device. The antibody or antibody fragment may regulate the adhesion of circulating endothelial progenitor cells and / or endothelial It can specifically recognize and bind to epithelial cell surface antigens, resulting in cells adhering to the surface of the device. The cell surface antigens are vascular endothelial growth factor receptors 1, 2, and 3 (VEGFR- 1, VEGFR-2, and VEGFR-3, as well as VEGFR receptor family members isoform), Tie-1, Tie2, CD34, Thy-1, Thy-2, Muc-1 8 (CD146), CD30, stem cell antigen 1 (Sca-1), stem cell factor (SCF or c-Kit ligand), CD133 antigen, VE-cadherin, P1H12, TEK, CD 31, Ang-1, Ang-2, or on the surface of endothelial progenitor cells and / or endothelial cells In one embodiment, a single type of antibody that reacts with one antigen can be used. Alternatively, antibodies and / or antibody fragments directed against different cell surface antigens may be used. Multiple different types of antibodies and / or antibody fragments directed against the In one embodiment, anti-CD34 and anti-CD133 antibodies and / or antibody fragments are used in combination. .

[0122] As used herein, a "therapeutically effective amount of an antibody or antibody fragment" refers to the amount of endothelial progenitor cells and The term "antibody" refers to the amount of antibody that promotes adhesion of endothelial cells and / or endothelial cells.

[0123] Antibodies and / or antibody fragments are used to identify the antigens to which they bind. To ensure accessibility, the compound may be immobilized on the medical device in an oriented manner. , at least 1%, at least 3%, at least 5%, at least 8%, at least 10 %, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, at least 45%, at least 50%, at least 6 0%, at least 70%, at least 80%, or at least 90% of the antibodies and / or or antibody fragments are available to bind to cell surface antigens. or at least 1%, at least 3%, at least 5%, at least 8%, or at least at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, At least 60%, at least 70%, at least 80%, or at least 90% anti- The original binding sites are not blocked or denatured. For example, the at least 1%, at least 3%, at least 5%, at least 8%, at least 10%, At least 15%, at least 20%, at least 25%, at least 30%, at least At least 35%, at least 40%, at least 45%, at least 50%, at least 60% , at least 70%, at least 80%, or at least 90% of the Fab region is completely exposed and available for antigen binding.

[0124] For example, antibodies and / or antibody fragments may have an Fc domain anchored and an antigen-binding Fab domain. The main can be immobilized on the medical device in an oriented manner with the main fully exposed. In embodiments, the majority of antibodies have at least one N-linked carbohydrate in the Fc region of their heavy chains. Therefore, immobilization strategies have been developed that utilize the Fc domain to introduce novel reactive moieties into the antibody structure. For example, two types of oligosaccharide modifications that can be used to modify antibodies are The first type utilizes the oligonucleotides found in the Fc region to provide reactive aldehyde groups. accompanied by sugar oxidation 103、104 After oxidation, the newly formed aldehyde moiety is converted to an amine residue. May be covalently bonded to the end surface 105、106 Mutant β1,4 galactosyltransferase Another technique uses the enzyme acetylase to replace the natural acetylglucosamine residue with a modified sugar. Modified sugars contain unique chemical groups incorporated into their molecular structure, often ketones or azides. The incorporation of modified sugars can be used to immobilize antibodies. c specific target is introduced. In the case of an azide moiety, the antibody undergoes a catalyst-free "click" cycloaddition. It may be covalently attached to a surface bearing cyclooctyne in an oriented manner via a reaction. By specifically modifying the Fc region of the antibody, both of these techniques allow the Fab region to It provides covalent immobilization of exposed antibodies (Figure 1).

[0125] In one embodiment, an oxidation method is used to immobilize the antibody or antibody fragment. Boc-hydrazide-PEG-amine (Quanta Biodesign) was used as a substrate or The PDA is immobilized on the PDA-coated surface of a medical device. The polydopamine-coated substrates / medical devices were then soaked in PBS / DMSO. The substrate / medical device is then exposed to oc-hydrazide-PEG-amine. Rinse, sonicate for 15 min in methanol, rinse with acetone, and dry under a stream of nitrogen. After successful immobilization of PEG chains, the modified surface was treated with trifluoroacetic acid (TFA) in methylene chloride. FA), followed by rinsing with ammonium hydroxide and The t-Boc protecting group was removed to form a hydrazide-rich surface for additional immobilization. The antibody or antibody fragment is oxidized to create the required aldehyde moiety (e.g., (In the Fc region of the antibody.) The antibody or antibody fragment is dissolved in a buffer solution (e.g., PBS). Sodium metaperiodate is added to the antibody solution to allow the reaction to proceed. After oxidation, the remaining metaperiodate is removed. Sodium iodate is removed using a desalting column (e.g., Sephadex G-25). The PEG-functionalized material is then immersed in the oxidized antibody solution and allowed to react. Sodium boron was added to reduce the cytoplasmic barrier formed between the antibody and the hydrazide-rich coating. Stabilizes the base.

[0126] The oxidation of the antibody or antibody fragment may be carried out at a suitable pH, for example, from about pH 3 to about pH 7, from about pH 3.5 to about pH 7. Approximately pH 6.8, approximately pH 4 to approximately pH 6.5, approximately 4.5 to approximately pH 6, approximately pH 5 to approximately pH 6, approximately 4 to about pH 6, about pH 5, about pH 5.5, about pH 5.6, about pH 5.8, or about It may be carried out at pH 6.

[0127] In another embodiment, enzymatic methods are used to immobilize the antibody or antibody fragment. Amino-PEG4-DBCO was attached to the PDA-coated surface of the substrate or medical device. For example, freshly prepared polydopamine coated substrates / medical The device is exposed to amino-PEG-dibenzocyclooctyne in PBS. The medical device was rinsed with acetone, sonicated in methanol for 15 minutes, and then rinsed with acetone. , and dry under a stream of nitrogen.

[0128] To functionalize an antibody or antibody fragment, a DBCO-reactive moiety is added, for example, to the Fc Biomolecules (e.g., antibodies) may be modified using enzymatic methods to create biomolecules. Remove the DBCO-reactive moiety away from the active site of the molecule (e.g., in the Fc region of an antibody). Step 1 involves removing terminal groups from a biomolecule (e.g., an antibody or antibody fragment). This may involve removing lactose residues (e.g., using β-1,4-galactosidase). β-1,4-galactosidase catalyzes the cleavage of β1 from oligosaccharides. Highly specific exoglycosides that catalyze the hydrolysis of -4-linked D-galactopyranosyl residues This particular residue can be present in the Fc region of some antibodies. After removing the lactose sugar, the biomolecule (e.g., antibody or antibody fragment) is ligated to UDP-Gal In combination with NAz, an azide moiety can be introduced. For example, step 2 can be These may include incorporating alNAz (e.g., Gal-T(Y289L), UDP-G alNAz, 37°C, 16 hours). In one embodiment, the antibody or antibody fragment is Click-IT® GlcNAc Enzymatic L according to the instructions. Using the abeling System (Life Technologies), Briefly, antibodies or antibody fragments can be attached to P30 resin (Bio Pretreated buffer was separated using a microspin column prepared with 1.5 mL total volume (Rad). The antibody or antibody fragment is then added to the pretreated column and centrifuged. The resulting antibody solution was supplemented with β-1,4-galactosidase and incubated at 37°C. Buffer exchange of the sample into Tris-buffered saline (TBS) using P30 resin. After buffer exchange, the antibody solution was diluted with U Combined with DP-GalNAz, MnCl2, and Gal-T(Y289L), 30 Incubate at 37°C. After modification, the antibody or antibody fragment is buffer exchanged into PBS. Then, the DBCO-coated substrate or medical device is immersed in the antibody solution, and then P Wash with BS to remove physically attached antibodies.

[0129] In yet another embodiment, UV immobilization techniques are used to immobilize antibodies or antibody fragments. It utilizes indole-3-butyric acid-PEG to detect isotype-agnostic antibodies. It binds to the antibody via a conserved nucleotide binding site found on virtually all antibodies. Binding antibody or antibody fragment 183 .

[0130] antibody fragment Antibodies can be full length or formed from antibody fragments such as Fab, F(a) b')2, Fab', F(ab)', Fv, single-chain Fv (scFv), bivalent scFv (bi -scFv), trivalent scFv (tri-scFv), Fd, dAb fragments (e.g., War d et al., Nature, 341:544-546 (1989)), isolated CDR , bispecific antibodies, trispecific antibodies, tetraspecific antibodies, linear antibodies, single chain antibody molecules, and One of the antibodies (also known as multispecific antibodies) having an antigen-binding portion, including but not limited to multispecific antibodies. The antibody fragments may be linked using recombinant or synthetic linkers. Also encompassed by the present disclosure are single chain antibodies produced by the method described in Bird et al., Sci. ence, 1988, 242:423-426. Huston et al.,Proc. .Natl.Acad.Sci.USA,1988,85:5879-5883.

[0131] Papain digestion of antibodies produces fragments called "Fab" fragments, each of which contains a single antigen-binding site. Two identical antigen-binding fragments are produced, and their names reflect their ability to readily crystallize. Pepsin treatment generates the remaining "Fc" fragment, which still contains two antigen-binding sites. F(ab')2 fragments are generated that are capable of cross-linking antigens.

[0132] Fv is the minimum antibody fragment that contains a complete antigen-binding site. A chain Fv species contains one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Single-chain Fv (scFv) species consist of a dimer of one heavy chain variable domain and one The light chain variable domains of the Fv gene are arranged in a "dimer" fashion, similar to that in a two-chain Fv species, where the light and heavy chains are in a "dimer" fashion. can be covalently linked by a flexible peptide linker so that they can be linked in the " In this configuration, the three CDRs of each variable domain interact to form a V H -V L Dimer Table Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or a fragment containing only three CDRs specific for an antigen) Even half of the Fv (including the entire binding site) can recognize and bind to the antigen, albeit with lower affinity than the entire binding site. There is the ability to do so.

[0133] The Fab fragment contains the heavy and light chain variable domains, the constant domain of the light chain and the constant domain of the heavy chain. The Fab' fragment also contains the first constant domain (CH1) from the antibody hinge region. Several cysteines at the carboxyl terminus of the heavy chain CH1 domain Fab'-SH fragments differ from Fab fragments by the addition of additional residues from the constant domains (one or more The name Fab' is used to designate a cysteine residue (or residues) bearing a free thiol group. Fab' antibody fragments originally consist of pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0134] Single-chain Fv or scFv antibody fragments are fragments of the V of an antibody. H and V L domains, and these The domains are present in a single polypeptide chain. Generally, an scFv polypeptide is V, which enables the Fv to form the desired structure for antigen binding H Domains and V L Domain For a review of scFvs, see, e.g., Plu ckthun,in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. See 269-315.

[0135] A "bispecific antibody" is an antibody fragment that has two antigen-binding sites; The same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) bound to the heavy chain variable Domain (V H) that is too short to allow pairing between two domains on the same chain. By using a linker that allows the domains to pair with complementary domains on another strand, Bispecific antibodies are bivalent or bispecific antibodies. Bispecific antibodies can be used in a variety of applications, including, for example, those described in European Patent No. 404,097, PCT Application No. PCT / US93 / 01997, and PCT Application No. PCT / US93 / 01997. Publication WO 1993 / 01161, Hudson et al., Nat. Me d.9:129-34,2003, and Hollinger et al.,Proc More fully described in Natl. Acad. Sci. USA 90:6444-8, 1993. Triabodies and tetrabodies are also described in Hudson et al., It is described in Nat. Med. 9:129-34, 2003.

[0136] Antibody fragments can be produced by conventional means, such as enzymatic digestion, or by recombinant techniques. In certain situations, there are advantages to using antibody fragments rather than whole antibodies. The small size of the fragments makes them This allows for rapid clearance and may lead to improved access to solid tumors. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:12 9-134,2003.

[0137] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been generated by immunohistochemistry. obtained via proteolytic digestion of intact antibodies (e.g., Morimoto et al. al.,J.Biochem.Biophys.Methods 24:107-17, 1992, and Brennan et al., Science 229:81-3, However, these fragments are currently not readily expressed in recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be produced directly by E. coli. These fragments can be expressed in and secreted from E. coli and used to produce large quantities of Antibody fragments can be easily produced by isolating them from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically purified. to form F(ab')2 fragments (Carter et al., Biol. / Technology 10:163-7, 1992). Another method is to use F(ab') The two fragments are isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives containing a hydroxyl group are disclosed in U.S. Pat. Other techniques for the production of antibody fragments are known to the skilled practitioner. It will be clear to

[0138] The antibody or antibody fragment may comprise (a) an IgG constant domain, (b) an IgA constant domain, etc. It may comprise at least one constant domain of

[0139] IgG (e.g. IgG1, IgG2, IgG3, IgG4), IgM, IgA (Ig All antibody isotypes are encompassed by this disclosure, including IgA1, IgA2, IgD, or IgE. The antibody or antibody fragment may be a mammalian (e.g., murine, human) antibody or antibody fragment. The light chains of the antibody can be of type kappa or lambda. The alternative antibodies may comprise sequences from multiple immunoglobulin classes or isotypes, and may be used interchangeably. Selection of specific constant domains to optimize desired effector function is within the skill of the art. This is within the ordinary skill of the art.

[0140] The antibodies or antibody fragments of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific or bispecific antibodies or fragments thereof can bind to one target polypeptide. The antibodies can be specific for different epitopes (e.g., cell surface antigens) of the same virus, or multiple antibodies can be used. Antigen binding domains specific for a number of target polypeptides (e.g., cell surface antigens and other antigens) The antibody may contain multiple antigen-binding domains (specific for a single antigen, or specific for multiple cell surface antigens). In one embodiment, a multispecific antibody or antibody fragment has at least two different variable domains. and wherein each variable domain is directed to a distinct antigen or to a different epitope on the same antigen. Tutt et al., 1991, J. Immunol. ol.147:60-69. Kufer et al.,2004,Trends Bi otechnol. 22:238-244. The antibody may be coupled to another functional molecule, e.g., another peptide. The peptide or protein may be bound to or co-expressed with the peptide or protein. The antibody or antibody fragment may be attached to the antibody by any suitable method (e.g., chemical coupling, genetic fusion, non-covalent bonding, etc.). operably linked to one or more other molecular entities, such as another antibody or antibody fragment) can be combined to generate bispecific or multispecific antibodies having a second binding specificity. The present disclosure includes bispecific antibodies, wherein one arm of the immunoglobulin binds to a cell surface. The other arm of the immunoglobulin is specific for a surface antigen, and the other arm of the immunoglobulin targets a second therapeutic target (e.g., a different The antibody may be specific for a specific cell surface antigen (e.g., a target cell surface antigen, or another antigen) or may be conjugated to a therapeutic moiety.

[0141] Antibody production In one embodiment, the antibody is a monoclonal antibody, as described by Kohler and Milst ein (Continuous cultures, incorporated herein by reference) of fused cells secreting antibody of pre defined specificity.Nature 265:495-497,1 975) or may be obtained from commercial sources. Endothelial cells are used to generate monoclonal antibodies directed against endothelial cell surface antigens. It can be used as an immunogen.

[0142] For example, monoclonal antibodies directed against endothelial cells have been shown to induce H The cells can be prepared by injecting UVECs or purified endothelial progenitor cells. The mice are sacrificed and spleen cells are obtained. The spleen cells are typically washed with a non-ionic detergent, e.g. By fusing with myeloma or lymphoma cells in the presence of polyethylene glycol The resulting cells, including the fused hybridomas, are grown in a selective medium such as HAT medium. The cells are grown in a suitable medium using limiting dilution conditions, and the surviving cells are expanded in such medium. The supernatant is then extracted with the desired specificity. , i.e., screening for monoclonal antibodies reactive with endothelial cell antigens. do.

[0143] Functional Group coating or substrate, and / or polydopamine, linker, and / or or ligands (antibodies or antibody fragments) are surface functionalized using known crosslinking agents. The crosslinking agent can be divinylbenzene, ethylene glycol dimethicone, Acrylate, Trimethylolpropane Trimethacrylate, N,N'-Methylene-bis- acrylamide, alkyl ether, sugar, peptide, DNA fragment, or other known functionality Ligands include, but are not limited to, substantially equivalent agents. Ligands include, for example, carbodiimides. , carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur acids The present invention may be applied to the production of fluorinated compounds, such as nitrates, nitroxides, halides, or other suitable compounds known in the art. The compound may be attached to the coating or substrate by a coupling reaction involving the use of a compound such as U.S. Pat. No. 6,268,222.

[0144] the coating or surface of the substrate, and / or the polydopamine, linker, and and / or the ligand (antibody or antibody fragment) is modified to include at least one functional group. The organic polymer (e.g., PEG) can be modified to incorporate at least one Functional groups can be incorporated. For example, the functional groups can be maleimide or N-hydroxyl groups. The incorporation of functional groups allows for the formation of various linkers. The nanoparticles may be coated with a protective agent, such as a dendritic cell, or a dendritic cell, and / or a pharmaceutical / therapeutic agent.

[0145] Click Chemistry Because the coatings or substrates of the present invention can readily accommodate a wide range of ligands, The surface of the coating or substrate may be modified to incorporate functional groups. The substrate may also be modified with an organic polymer (e.g., PEG) that can incorporate functional groups. Alternatively, the ligand or therapeutic agent may be attached to a coating or substrate, or may be attached to a substrate under suitable conditions. The resulting polymer possesses functional groups capable of reacting with functional groups on PEG coated or attached to a substrate. Therefore, any ligand or therapeutic with a reactive functional group can be modified to incorporate the Drugs can be easily attached to the coating or substrate. This generalizable approach is described herein. This is called "click chemistry" in the literature and allows for a great deal of versatility. Any suitable reaction may be used as long as facile and controlled attachment of the ligand to the substrate or substrate can be achieved. The reaction mechanism can be adapted to "click chemistry." In one embodiment, the free triple bond is , which is introduced onto PEG that is already covalently attached to a coating or substrate. The bond is introduced to the desired ligand. The PEGylated coating or substrate and the ligand are When mixed in the presence of a copper catalyst, cycloaddition of the azide to the triple bond occurs, This results in the binding of the ligand to the coating or substrate. The hydroxyl and thiol functional groups are introduced into the coating or substrate and the desired ligand. wherein the coating or substrate has a maleimide functional group and the ligand has a thiol group; The reverse is also true: the double bond of maleimide reacts readily with thiol groups to form stable carbon atoms. In a third embodiment, an activated ester functional group, e.g., a succinyl group, forms a sulfur bond. The animidyl ester group and the amine group are linked to the coating or substrate and the desired ligand. Activated ester groups can be readily introduced into amine groups to form stable carbon-nitrogen bonds. Forms an amide bond.

[0146] medical devices A medical device is a device that is temporarily or permanently administered to a mammal for the prevention or treatment of a medical condition. These devices can be subcutaneously, percutaneously, or surgically introduced. and placed within an organ, tissue, or organ lumen, such as an artery, vein, or ventricle or atrium of the heart. Medical devices include any of the following: stents, stent grafts, polytetrafluoroethylene Ethylene (PTFE), Expanded Polytetrafluoroethylene (ePTFE), Coating Uncovered stents or covered stents, such as those covered with artificial vascular grafts Fixing devices for connecting artificial heart valves, artificial hearts and prosthetic organs to the vascular circulation Fixtures, artificial venous valves, abdominal aortic aneurysm (AAA) grafts, inferior vena cava filters, permanent drug infusion catheters, embolic coils, embolic materials used in vascular embolization (e.g., cross-linked PVA hydrochloride) rogel), vascular substitutes, vascular sutures, vascular anastomosis fixation devices, transmyocardial revascularization stents and / or or other conduits.

[0147] The medical device can be any device that can be implanted in a patient. For example, in one embodiment In some cases, the devices include stents, stent grafts, heart valves, catheters, and vascular prosthetic filters. - artificial hearts, external and internal left ventricular assist devices (LVADs), artificial vascular grafts, etc. For insertion into the lumen of a blood vessel or hollow organ.

[0148] A medical device is any device used for implantation in an organ or body part that contains a lumen. The medical device may be implanted in the lumen of an organ or blood vessel. Stents, stent grafts, synthetic vascular grafts, heart valves, catheters, vascular prosthetic fillers pacemakers, pacemaker leads, defibrillators, patent foramen ovale (PFO) septal closures Chain devices, vascular clips, vascular aneurysm occluders, hemodialysis grafts, hemodialysis catheters valves, atrioventricular shunts, aortic aneurysm graft devices or components, venous valves, sensors, sutures Sutures, vascular anastomosis clips, indwelling venous or arterial catheters, vascular sheaths, and drug delivery ports The present invention is not limited to the above.

[0149] Medical devices include patent foramen ovale (PFO) closure devices, circulatory assist devices (e.g., left ventricular assist devices), LVAD, Extracorporeal Membrane Oxygen Membrane Adsorption (ECMO), Neurovascular Clip, Artificial Joint, Vena Cava The device may be a filter, a component of an artificial heart, etc.

[0150] A stent, when inserted or implanted in the lumen of a blood vessel, can expand the cross-sectional lumen of the blood vessel. The stent may be any medical device that can be used for the treatment of a disease. The stent may be a coated stent, such as one coated with PTFE or ePTFE. In one embodiment, the stent is used to treat coronary artery blockages or to treat splenic, carotid, or It is delivered percutaneously to seal dissections or aneurysms of the venous, iliac, and popliteal vessels. In embodiments, the stent is delivered to a venous vessel. The stent may comprise a polymer and / or The stent may include a metallic structural element. Aluminum, gold, platinum-iridium, or Elgiloy and MP35N and other ferrous materials The stent may be placed on the catheter at the treatment site where the stent is released from the catheter. The stent can be delivered through a body lumen of a blood vessel and expanded until it comes into direct contact with the luminal wall of the blood vessel. Stents include metallic coronary stents, metallic peripheral arterial stents, and bioabsorbable peripheral stents. stents, and bioabsorbable coronary stents, but are not limited to.

[0151] The synthetic graft can be any artificial prosthesis that has biocompatible characteristics. In one embodiment, the synthetic graft is made from polyethylene or polytetrafluoroethylene. In another embodiment, the synthetic graft can be made of polyurethane, cross-linked PVA hydrogel. In a further third embodiment, the biocompatible foam comprises a synthetic graft, a hydrogel, or a mixture thereof. The raft is made of meshed polycarbonate with a urethane inner layer and meshed polyethylene The synthetic graft includes an outer layer of terephthalate. , side-to-end, side-to-side or intraluminally, and in vascular anastomoses, or may be used to bypass diseased vessel segments, for example, as an abdominal aortic aneurysm device. .

[0152] The artificial valve may be a prosthetic heart valve or a prosthetic venous valve. The artificial valve may be a prosthetic aortic valve. Prosthetic heart valves (artificial heart valves) can be used for various purposes, including artificial pulmonary valves, artificial mitral valves, and artificial tricuspid valves. Transcatheter aortic valve (TAVR), transcatheter mitral valve, transcatheter tricuspid valve valves, surgically implanted bioprosthetic aortic valves, surgically implanted bioprosthetic mitral valves, surgically implanted metallic mitral valves, and surgically implanted metallic aortic valves, Not limited to these.

[0153] Vascular replacements include endovascular aneurysm repair (or endovascular aortic repair) (EVAR), and and ePTFE bypass graft materials.

[0154] Medical devices include mitral valve clips, tricuspid valve clips, atrial appendage closure devices, and pacemaker leads. , Automatic Implantable Cardioverter Defibrillator (AICD) leads, pacemaker boxes, and automatic implantable cardioverter defibrillators Coronary medical devices, including but not limited to implantable cardioverter defibrillator (AICD) boxes It can be said that:

[0155] Medical devices have a luminal surface (or blood-contacting surface) and an external surface (or abluminal surface). The coatings of the present invention may have a luminal surface (or a blood-contacting surface). surface), and / or the external surface (or abluminal or tissue-contacting surface) .

[0156] This coating on medical devices is intended to form a monolayer (confluent or may stimulate the development of subconfluent (sub-confluent) and / or localized chronic inflammatory responses and Other thromboembolic complications resulting from vascular injury during implantation of medical devices may be modulated.

[0157] Medical devices can be made from a number of materials. Medical devices can be made from stainless steel, Nitinol , MP35N, gold, tantalum, platinum or platinum iridium, or carbon or carbon fiber Other biocompatible metals and / or alloys such as cellulose acetate, cellulose nitrate, Polycone, cross-linked polyvinyl acetate (PVA) hydrogel, cross-linked PVA hydrogel foam, polyurethane styrene, polyamide, styrene isobutylene-styrene block copolymer (Krato n), polyethylene terephthalate, polyurethane, polyamide, polyester, polyamide Polyester, polyanhydride, polyethersulfone, polycarbonate, polypropylene Polyethylene, high molecular weight polyethylene, polytetrafluoroethylene, or other biocompatible polymers -materials or their derivatives such as polylactic acid, polyglycolic acid or their copolymers Blend of copolymer polyester, polyanhydride, polycaprolactone, polyhydroxyvalerate hydroxybutyrate or other biodegradable polymers, or mixtures or copolymers, extracellular matrix a drug component, protein, collagen, fibrin or other bioactive agent, or a mixture thereof.

[0158] For example, stents may be made of stainless steel, Nitinol (NiTi), or chromium alloys. and may be made of a biodegradable material. In one embodiment, the stent is made of a biodegradable material. Synthetic vascular grafts may be made of cross-linked PVA hydrogel, polytetrafluoroethylene, or Polytetrafluoroethylene (PTFE), Expanded Polytetrafluoroethylene (ePTFE), High-density Porous High-density polyethylene (HDPE), polyurethane, and polyethylene terephthalate, The present invention is based on biodegradable materials such as polylactide polymers and polyglycolide polymers or their It may also be made of a copolymer of

[0159] In one embodiment, the medical device may be a preserved blood vessel that has been denuded or stripped of cells. The preserved blood vessels can be derived from human, porcine or bovine sources. For example, it forms a scaffold suitable for use as a vascular graft segment.

[0160] The method can be for treating a mammal with a vascular disease, The method involves implanting the coated medical device into the organ or blood vessel of a patient. Once in vivo, the endothelial progenitor cells are stimulated by the antibodies or antibody fragments present on the coating. and / or by recognition and binding of cell surface antigens on endothelial cells, endothelial progenitor cells and and / or endothelial cells are captured on the surface of the coated medical device. Once the progenitor cells and / or endothelial cells attach to the medical device, they proliferate and differentiate, Formation of confluent or subconfluent functional endothelium on the blood-contacting surface of a medical device Alternatively, or in addition, the medical device may be endothelialized in vitro prior to implantation of the medical device. The membrane is coated with endothelial progenitor cells and / or endothelial cells. The cells are progenitor, stem, and / or endothelial cells isolated from the patient's blood, bone marrow, or vasculature. The presence of endothelial cells on the blood-contacting surfaces of medical devices may be due to excessive It may inhibit or reduce intimal hyperplasia and / or thrombosis.

[0161] Human umbilical vein endothelial cells (HUVECs) were cultured as described in Jaffe et al., J. Med., 1999, pp. 111-114, which are incorporated herein by reference. ,et al.,J.Clin.Invest.,52:2745-2757,1973 Briefly, cells can be obtained from umbilical cords by treatment with collagenase. Removed from the vessel wall by low endotoxin fetal bovine serum, preservative-free porcine M199 containing heparin, endothelial cell growth supplement (ECGS), and glutamine They are grown in gelatin-coated tissue culture flasks in medium.

[0162] Endothelial progenitor cells (EPCs) were isolated using the method described by Asahara et al. (Isolation of EPCs) f putative progenitor endothelial cells for angiogenesis.Science 275:964-967,199 7, incorporated herein by reference) from human peripheral blood. Briefly, magnetic beads coated with antibodies against CD34 were used to fractionate the cells. Incubate with human peripheral blood. After incubation, the bound cells are eluted. Alternatively, enriched media isolation can be used to isolate these cells. Briefly, peripheral venous blood was collected from healthy male volunteers, The mononuclear cell fraction was isolated by density gradient centrifugation, and the cells were supplemented with fetal bovine serum, human VEGF- A, human fibroblast growth factor-2, human epidermal growth factor, insulin-like growth factor-1, and Fibronectin in EC basal medium-2 (EBM-2) supplemented with ATP and ascorbic acid was The EPCs were grown for 7 days and the medium was changed every 48 hours. The cells express CD133, CD45, CD34, CD31, VEGFR-2, It can be characterized by fluorescent antibodies against Tie-2 and E-selectin.

[0163] Conditions to be treated / prevented The present disclosure provides a method for treating one or more symptoms associated with various diseases / conditions using the present medical device. Treat, prevent (or prophylactically treat), eradicate or reverse The conditions to be treated or prevented include restenosis, atherosclerosis, thrombosis, and the like. vascular diseases such as vascular obstruction (e.g., resulting from thrombosis), aneurysms, and coronary artery disease; cancer; In one embodiment, the vascular wall is treated with a stem cell. artificial or synthetic vascular grafts, heart valves, abdominal aortic aneurysm devices and their components and for establishing vascular homeostasis, thereby Methods are provided for preventing excessive intimal hyperplasia, such as in restenosis.

[0164] This medical device induces smooth muscle cell migration along the luminal surface at the implantation site of the medical device, smooth muscle Tissue-based cellular differentiation and / or collagen deposition may be reduced or inhibited. This may reduce or inhibit excessive intimal hyperplasia and restenosis of the vascular graft.

[0165] The medical devices and methods may be used in any blood vessel, such as any artery or vein. any of the following, including the aorta, infrainguinal artery, aortoiliac artery, subclavian artery, mesenteric artery, and renal artery The medical devices and methods are also applicable to peripheral arteries such as the femoral artery. Other types of vascular occlusions, such as those resulting from dissecting aneurysms, may also be treated with the present disclosure. The medical devices and methods may be used in any duct or cavity in a mammal. Subjects that may be treated using the stents and devices of the present invention include humans, horses, dogs, These are mammals, including cats, pigs, rodents, and monkeys.

[0166] The present disclosure relates to a method for treating a mammalian pulmonary artery disease, including implanting a medical device into the lumen of a blood vessel or tubular organ of the mammal.

[0013] A method for treating vascular disease in a mammal is provided, wherein the medical device comprises a It is coated so that it can be used.

[0167] The present disclosure provides methods for recruiting cells to blood-contacting surfaces of medical devices in vivo. In one embodiment, the method includes implanting a medical device into a blood vessel of the subject. The method comprises the steps of: providing a blood-contacting surface configured to bind target cells circulating in the blood of a subject; Target cells attached to the blood-contacting surface proliferate and form a functional endothelium in situ. or self-endothelializing the surface of the device as it restores normal endothelium at the site of vascular injury. In one embodiment, the medical device can be biodegradable, or biodegradable, biocompatible. In this embodiment, when implanted in a blood vessel, the biodegradable medical device The device may undergo in situ degradation, and the neoendothelium formed on the luminal surface of the device may form functional neoendothelium. Restore vascular continuity through the injured area to form blood vessels.

[0168] Intimal hyperplasia is an undesired progression of smooth muscle cell proliferation and / or matrix deposition in the vascular wall. As used herein, "restenosis" refers to a recurrent narrowing of the lumen of a blood vessel. The blood vessel may become occluded due to restenosis. After PTCA or PTA, the intima usually Smooth muscle cells from the tunica media and adventitia, which are not present in the intima, proliferate and migrate to the intima, where they secrete proteins. secrete and form accumulations of smooth muscle cells and matrix proteins within the intima. , causing narrowing of the arterial lumen and reducing blood flow distal to the narrowing. "Inhibiting or reducing restenosis" means preventing restenosis and complications arising therefrom. Inhibition or reduction of smooth muscle cell migration and / or proliferation accompanied by prevention of protein secretion vinegar.

[0169] This medical device is intended to be used to achieve a therapeutic benefit ("treat") or prophylactically. It can be administered to a subject (e.g., implanted in a subject) to achieve a benefit ("prevent"). Therapeutic benefit may include eradication or reversal of the condition being treated and / or a reduction in the severity of a condition related to the condition. A prophylactic benefit means the eradication or amelioration of one or more symptoms associated with a condition. prevention or delay of the onset of, and / or the onset of, one or more symptoms associated with the condition In some embodiments, administration (e.g., implantation) of the medical device prevents the condition from developing or worsening into a more serious condition.

[0170] "Treating" or "treatment" of a condition, disorder, or condition means (1) treating the condition, disorder, or condition; may be suffering from or predisposed to a condition, but may not be affected by any circumstances, disorders or is a condition, disorder, or state occurring in a subject who has not yet experienced or shown clinical symptoms of the condition. (2) preventing or delaying the onset of clinical symptoms of a condition, disorder, or state suppression of the disease, i.e., the occurrence or recurrence of the disease (in the case of maintenance treatment), or Cessation or reduction of at least one clinical symptom, sign, or test of or (3) to alleviate the disease, i.e., to reduce the severity of the condition, disorder, or if or regression of at least one of the conditions, or its clinical or subclinical symptoms or signs This includes causing

[0171] Pharmaceutical substances The coating of the device may include one or more medicinal substances. inhibits cell migration and / or proliferation, inhibits or reduces thrombus formation, and promotes endothelial cell proliferation and and / or inhibit or reduce restenosis after implantation of a medical device. The medicinal substance acts downstream of the device to affect vascular properties or to induce solid organ damage. Medical devices can have local and / or systemic effects (e.g., distal to the device). ) can be demonstrated.

[0172] The pharmaceutical substance is a vasodilator (prostacyclin (PGI2), calcitonin gene-related The compound may be a peptide (such as α-CGRP).

[0173] Pharmaceutical substances are effective in treating vascular diseases such as atherosclerosis and restenosis. For example, the pharmaceutical agent may be a cytotoxic or cytostatic agent, an antiproliferative agent, an antitumor agent, Antibiotics / antimicrobials, antioxidants, endothelial growth factors, thrombin inhibitors, immunosuppressants, antihemoglobins Platelet aggregators, collagen synthesis inhibitors, therapeutic antibodies, nitric oxide donors, antisense oligonucleotides oligonucleotides, wound healing agents, therapeutic gene transfer constructs, peptides, proteins, extracellular Matrix components, vasodilators, thrombolytics, antimetabolites, growth factor agonists anti-mitotics, statins, steroids, steroidal and non-steroidal anti-inflammatory drugs, Angiotensin-converting enzyme (ACE) inhibitors, free radical scavengers, PPAR Anti-cancer chemotherapeutic agents include, but are not limited to, vasopressin agonists, aromatase inhibitors, and other anti-cancer chemotherapeutic agents. Some of the aforementioned pharmaceutical substances are, for example, cyclosporine A (CSA), rapamycin, rapamycin derivatives, mycophenolic acid (MPA), retinoic acid, n-butyric acid, butyric acid Derivatives, Vitamin E, Probucol, L-Arginine-L-Glutamate, Everolimus , Sirolimus, Biolimus, Biolimus A-9, Paclitaxel, Puerarin, Platelets Factor 4, basic fibroblast growth factor (bFGF), fibronectin, simvastatin, Fluvastatin, dihydroepiandrosterone (DHEA) and 17 beta-estradiol Contains radiol.

[0174] Examples of pharmaceutical agents that can be incorporated into the coating include prostacyclin, prostacyclin, Phosphorus analog, alpha-CGRP, alpha-CGRP analog, or alpha-CGRP Receptor agonist; prazosin; monocyte chemoreactive protein-1 (MCP-1); rapamycin immunosuppressants such as isoflurane, drugs that inhibit smooth muscle cell migration and / or proliferation, thrombin antithrombotic agents such as thrombin inhibitors, immunomodulators such as platelet factor 4 and CXC chemokines; Inhibitor of the 3CR1 receptor family; anti-inflammatory drug, dihydroepiandrosterone (DHE) A) Steroids such as testosterone and estrogens such as 17beta-estradiol statins such as simvastatin and fluvastatin; fenofibrate and Other lipid-lowering drugs, such as PPAR-alpha ligands, PPAR-degrading drugs, such as rosglitazone, ATP and PPAR-gamma agonists; nuclear factors such as NF-κB and collagen synthesis inhibitors such as acetylcholine, adenosine, 5-hydroxytryptamine or serotonin Vasodilators, substance P, adrenomedullin, basic fibroblast growth factor (bFG) F), platelet-derived growth factor (PDGF), endothelial growth factor (EGF), vascular endothelial cell proliferation Growth factors that induce proliferation and differentiation of endothelial cells, such as VEGF; Protein tyrosine kinase inhibitors or anti-angiogenesis inhibitors such as linib and imatinib Compounds; peptides or antibodies that inhibit mature leukocyte adhesion, antibiotics / antimicrobials, and tachykinins Others such as kinin, neurokinin or sialokinin, tachykinin NK receptor agonists PDGF receptor inhibitors such as MLN-518 and its derivatives, butyric acid and Derivatives puerarin, fibronectin, erythropoietin, darbepotin, serine protein The aforementioned pharmaceutical substances include, but are not limited to, steroid enzyme protease-1 (SERP-1), and the like. , applied alone or in combination and / or mixtures thereof to the coating on the equipment It can be done.

[0175] Prostacyclin (PGI2) binds to specific G protein-coupled receptors, the IP receptors. and / or binds to the nuclear receptor, peroxisome proliferator-activated receptor (PPAR) δ After its synthesis and release, prostacyclin is an autocrine and paracrine mediator. Cyclins exert local anticoagulant and vasodilator properties and are not preserved, but are instead inactive substitutes. It is converted to its metabolite, 6-keto prostaglandin F1α (PGF1α), by a non-enzymatic process. Prostacyclin is rapidly converted by adenylyl cyclase / cyclic cyclase. It induces relaxation of vascular smooth muscle via the AMP transduction system and is effective in all blood vessels studied. Stable prostacyclin analogs are used in this coating and and methods.

[0176] Calcitonin gene-related peptide (α-CGRP) stimulates vascular endothelium in the absence of endothelium-derived NO. It can stimulate vasodilation, which can be mediated through the CGRP1 receptor.

[0177] The pharmaceutical agent may be locally released into adjacent or surrounding tissue in a sustained or controlled release manner Pharmaceutical substances can have a therapeutic effect locally and / or systemically.

[0178] Combination therapy The medical device may be administered alone or in combination with surgery, another medical device, and / or another therapeutic agent (e.g., administered / implanted in combination with one or more other therapies, such as a second therapeutic agent It can be rare.

[0179] Such combination therapy may be administered in combination with other drugs, such as steroids, antihistamines, or other anti-inflammatory drugs, to treat a variety of conditions, including: These may have additive or synergistic effects on the risk of recurrence (frequency of recurrence).

[0180] The medical device may be administered / implanted simultaneously with a second therapy. Alternatively, the second therapy may be administered before or after administration / implantation of the medical device.

[0181] In some embodiments, the second therapeutic agent is, for example, doxorubicin, paclitaxel. , melphalan, vinca alkaloids, methotrexate, mitomycin C or ethotrexate The cytotoxic agent can be a conventional chemotherapy drug such as benzodiazepine. 065 analogs, calicheamicin, maytansine, dolastatin analogs, rhizoxin, and powerful agents such as palytoxin may be used.

[0182] Subjects that may be treated using the medical devices, methods and compositions of the present invention are mammals. , including humans, horses, dogs, cats, pigs, rodents, monkeys, etc.

[0183] The following are examples of the disclosure and should not be construed as limiting.

[0184] [Example] Example 1: Preclinical evaluation of orthotopic prosthetic aortic valve implantation Animal models After approval by the Institutional Review Board, the experimental evaluation was carried out on six adult Yorkshire pigs (approximately 60 kg). The animals will be fasted for approximately 12 hours before induction of anesthesia. The patient was given an anesthetic cocktail containing ketamine, xylazine, and atropine via IM injection. The pig will be transported to the Vivarium pre-operative room. Anesthesia was administered using 5% isoflurane with 70% nitrous oxide / oxygen. Once anesthetized, an IV catheter will be inserted into the ear vein. Once IV access is achieved, the pig will be placed in the The pig will then be intubated and placed on a ventilator. The pig will then be transported to the operating room at Vivarium. The pigs were then transported to a veterinary ward where they were kept at approximately 2-3% isoflurane and 70% CO2 throughout the procedure. The surgical plane of anesthesia will be maintained with 100% nitrous oxide / oxygen and monitored (EKG) Once the animal is in the surgical plane of anesthesia (lack of jaw reflex), If the patient is in good physical condition (as confirmed by stable parameters on EKG), the surgical procedure will be performed. This becomes the case.

[0185] Surgical procedure Valve implantation is performed in a sterile environment with complete anesthesia, surgical, and angiographic equipment This setup was performed using a monoplane fluoroscopic angiography system (Siemens, Munic h, Germany) and a transthoracic echocardiographic console (GE E95s) Fluoroscopy, angiography, and echocardiography imaging of the aortic root during the procedure TH imaging will be performed prior to implantation to obtain optimal perpendicular views of the implant site. The distance of the left and right coronary ostia from the aortic annulus relative to the height of the V-frame will be determined. Additionally, a pigtail catheter was placed deep into the right coronary sinus to allow for the insertion of an aortic valve prosthesis. Assists positioning by providing reliable landmarks for accurate alignment of the This further encourages the placement of a pacemaker lead in the right ventricle.

[0186] Device The 23mm Edwards SAPIEN valve will be used, and three will be Three were provided from the same facility, and three were coated with the endothelial progenitor cell capture coating described herein. (e.g., polydopamine, or polydopamine and antibody, or polydopamine) Edwards SAPIEN 3 (coating containing PEG, PEG and antibody). The Transcatheter Heart Valve (THV) is balloon expandable. The vascular system is equipped with a radiopaque cobalt-chromium frame, a tricuspid bovine pericardial tissue valve, and a poly Ethylene terephthalate (PET) fabric skirt. Edwards C The omander delivery system is designed to facilitate valve alignment to the balloon, tracking and positioning of the THV. The handle contains a Flex Catheter that assists in positioning. Flex Wheel to control songs, and Balloon Lock and Fine Includes an Adjustment Wheel to aid in valve alignment within the native annulus. The balloon catheter has a defined working length. It has a radiopaque valve alignment marker that defines the valve. A transverse center marker is provided to assist in valve positioning. A radiopaque triple marker near the catheter guides the Flex Catheter's location during placement. Shows.

[0187] The 14F expandable introducer sheath is inserted into the common femoral artery for transfemoral approaches and into the subclavian artery for transfemoral approaches. The delivery catheter is surgically inserted into the subclavian artery. tz extra-stiff 0.035-inch guidewire (Cook, Inc., Bloomberg) The THV is inserted into the left ventricle via a catheter (Bangton, Indiana). The placement was determined by aortic root angiography using a pigtail catheter and transthoracic echocardiography ( The final placement position will be confirmed by aortic root angiography (TTE) guidance. The patient will be documented by radiography and TTE. Rapid pacing should then be initiated. When the systolic blood pressure drops below 50 mmHg, the balloon is inflated. When the inflation barrel is empty, the balloon is deflated. When the valve is fully deflated, the pacemaker will be turned off.

[0188] follow up On days 7 and 14, valve evaluation was performed by transthoracic echocardiography after induction of general anesthesia. After 14 days of echocardiographic evaluation, the animals were sacrificed by lethal injection and the prosthesis was removed. The TAVR valve was removed for gross examination and histological and scanning electron microscopic evaluation of the valve leaflets. Important parameters to evaluate are the presence of macroscopic and microscopic thrombi, and The degree of coverage of the valve leaflets by the endothelium will be included.

[0189] Procedural Considerations Aortic valve sizing in preclinical studies has been performed using the same sizing as is typically used in human clinical cases. A different strategy is needed. In the clinic, the valves to be replaced are those that are diseased and typically In animal models, they are healthy. Healthy annulus is malleable and tends to expand upon emergence from anesthesia, which is why animal models Dell valves must be appropriately oversized to avoid migration and stability issues However, if it is too large, the inconsistencies seen preclinically and reported in human clinical studies may be obscured. This can lead to an increase in other complications, such as fatal arrhythmias. It is important to note that the animals (and the annulus) grow during the study. If the valve dimensions are exceeded, significant paravalvular leakage may occur, leading to complications in the later stages of the study. possible.

[0190] -Mitigation strategies - Adult pigs (60 kg) were used in these studies to minimize annular growth. This will be possible.

[0191] -The study will be subacute (up to 14 days). The sheep model will have moderate to severe clinical signs during follow-up. Because of the significant weight and size increase, it is the more commonly selected and mismatched valve for chronic valve evaluation. Limit the risk of paravalvular leak due to the puncture.

[0192] Transcatheter delivery of prosthetic valves posed new challenges in animal models. Not only must the size and appropriate valve size be considered, but the vascular access and delivery methods must also be considered. The diameter of the peripheral vessel used must be of appropriate size. However, if the catheter profile is too large to fit into the peripheral vessels, Complications may occur or the valve may not be able to be delivered.

[0193] -Mitigation strategies - Adult pigs (60 kg) were used in these studies to minimize annular growth. This will be possible.

[0194] Annular size and peripheral arterial diameter, as well as the dimensions of the ascending aorta and the location of other vascular structures This also impacts the success of TAVR implantation and research. Length directly affects the success of higher profile implants.

[0195] -Mitigation strategies - In contrast to sheep, which tend to have brachiocephalic arteries arising from the ascending aorta In porcine brachiocephalic arteries, the aortic arch gives rise to the longer ascending aorta, and the implant Allows for proper fit into the annulus.

[0196] Left Main Coronary Artery Occlusion and Ultimately Heart Block Preclinical Study of TAVR in a Porcine Model Pigs have a coronary artery that begins near the aortic valve. This differs from humans, where the coronary arteries are located further away from the aortic annulus. In animal models, the distance between the aortic annulus and the coronary artery ostia is short, increasing the tendency for coronary artery ostia to become blocked. do.

[0197] Example 2: Universal coating method for oriented antibody immobilization on the surface of implantable materials Development of Objective: Surface endothelialization of implanted intravascular devices leads to rapid healing and reduced thrombogenicity. We have demonstrated that circulating endothelial progenitor cells can be captured to promote endothelialization. Developing a Genous Technology Dextran-Mediated Coating of Anti-Human CD34 Antibody on the Tent However, this method cannot coat other materials such as ePTFE. The goal of this study is to develop a method for immobilizing anti-CD34 antibodies on various surfaces. The aim was to develop a universal coating method.

[0198] Methods: Polydopamine membranes were applied to various materials, i.e., metal stents, ePTFE grafts, and and on the surface of pig pericardium under slightly basic conditions via oxidative self-polymerization of dopamine. Then, a polyethylene glycol (PEG) crosslinker was applied to form the This is bound to a polydopamine coating at one end and to the F of an antibody or antibody fragment at the other end. The coating layer was analyzed by profilometry, X-ray photoelectron spectroscopy, and The functionality of the CD34 antibody-coated surface was analyzed using a scanning electron microscope. was assessed by cell binding assay.

[0199] Results: CD34 antibody-coated surfaces of different materials bind CD34+ cells However, it did not bind CD34- cells. The surface functionalized with EG did not bind CD34+ cells. The coating layer thickness was , in the micrometer range, and the surface was uniform and smooth.

[0200] Conclusion: We have developed a universal coating method for oriented antibody immobilization, which is compatible with ePTFE coatings. Similar to rafts, they can be applied to reduce thrombosis in biological and mechanical valves.

[0201] Example 3: Surface modification of implantable materials for novel therapeutic applications the purpose: Develop a method for immobilizing biomolecules on a wide range of solid surfaces, and develop novel antibody functional groups. The effectiveness of the coating will be tested by developing a synthetic prosthesis.

[0202] The potential of antibody-functionalized materials as a new platform for localized drug delivery Demonstrate the following.

[0203] Research 1 We have developed a polydopamine-based polymer in combination with an appropriately functionalized polyethylene glycol layer. (PDA) surface modification has become a universal method for immobilizing bioactive molecules on a wide range of biomedical materials. Suppose a platform is created.

[0204] Background - Biomolecule Immobilization: Current immobilization techniques: Most standard biomaterials are made from inert substances that lack functional moieties for chemical bonding. Therefore, non-covalent physical adsorption is a commonly used method for immobilizing biomolecules. However, this technique can result in randomly distributed molecules, loss of biological activity, and This results in a coating that is easily removed from the surface of the material, resulting in more reliable results. Alternative methods provided include covalent immobilization through chemical, plasma, or gamma radiation treatment. These techniques involve the introduction of novel chemical moieties for the synthesis of fibronectin, collagen, Gelatin, and RGD 66 However, unfortunately, However, they still often result in randomly distributed, inactive molecules. Furthermore, these techniques have limited penetration depth and adversely affect the mechanical properties of the material. There is a possibility 67 However, it cannot be used universally on all substrates. A surface functionalization method that effectively covers the substrate, preserves the mechanical properties of the substrate, and is applicable to a wide range of materials. It is desirable to develop a law.

[0205] Polydopamine membrane: To effectively control surface properties, polymer coatings are used in many applications. It has been 68~70 Recently, thin nanotubes fabricated by sequential deposition of interacting polymers have been Polymer films, known as layer-by-layer (LbL) deposition, have been shown to have drug loading capabilities and biomolecules. They show promise as surface modifiers, offering desirable properties such as the possibility of modification. Most LbL deposition techniques suffer from the same problems as above and involve multiple steps and complicated initial processes. Surface modification is required.

[0206] A new form of LbL deposition, which has recently attracted considerable interest, takes advantage of the spontaneous formation of PDA films. These problems have been overcome by functionalizing the material using PDA. Synthetic hydroxybenzoates formed via oxidative self-polymerization of dopamine (DA) under very basic conditions. These films have the ability to form on virtually any solid surface. This unique property allows thin films to be deposited on a wide range of materials simply by immersing the substrate in a DA solution. The work by Lee et al. We have confirmed the presence of PDA films on a number of different materials after coating. Examples of these materials are metals, , glass, synthetic polymers (PTFE and PDMS) 71 25 after 3 hours of soaking X-ray photoelectron spectroscopy (XPS) of the material revealed the complete absence of substrate-specific signals. This suggests a cohesive coating thickness of at least 10 nm.

[0207] In addition to the universal and facile nature of the deposition process, PDA coatings are also highly reactive due to secondary reactions. The membrane has been found to be a very versatile platform for Under certain conditions (neutral pH and room temperature), thiols or thiols can be formed via Michael addition or Schiff base formation. In previous studies, PDA was functionalized with a coating containing a primary amine. Taking advantage of the reactivity of the coated substrate, thiolated polyethylene glycol (PEG), amino Monomerized PEG, trypsin, bovine serum albumin (BSA), concanavalin A, RNas e B, and some antibodies were immobilized. In most cases, biomolecules were directly immobilized. , and biological activity was maintained. 72 .

[0208] Polyethylene glycol crosslinker: The main factors affecting the biocompatibility of materials are contamination (non-specific proteins and cells) PEG is known for its excellent biocompatibility and antifouling properties. It is a hydrophilic polyether compound that has found wide application in both medicine and industry. 73 . hydrophilic Modification of surfaces with polymer chains reduces protein adsorption and dramatically reduces nonspecific cell adhesion. has been shown to reduce 74~77 Currently, physical adsorption, self-assembled monolayers, and chemical coupling are being investigated. Many techniques have been used to modify surfaces with PEG, including polymerization and grafting. are.

[0209] Studies examining the antifouling properties of PEG have shown that it effectively inhibits nonspecific binding to many substrates. Chen et al. showed that a PEG film can prevent the formation of PEG on the polyaniline surface. demonstrated that the surface showed a significant reduction in both protein adsorption and platelet adhesion. did 78 Zhang et al. reported that the PEG coating formed on SS was effective in preventing the formation of fibroblasts in bovine blood. It has been shown to be highly effective in preventing serum albumin and gamma globulin adsorption. did 79 PEG chains were used by Wang et al. to modify PTFE surfaces. The PEG-modified PTFE showed increased hydrophilicity and was very effective in preventing the adsorption of bovine serum albumin. has been shown to be effective in 80 .

[0210] In addition to its excellent antifouling properties, PEG can also be used as a cross-linking molecule for peptide modification. The ever-expanding inventory of functionalities allows PEG to be conjugated to virtually any biomolecule. Heterobifunctional PEG chains can be used to form hydrazides, azides, and cyclohexyl groups. The amine and thiol functional groups combined with octyl and biotin were used to create Due to the versatility and popularity of PEG chains for biomolecule modification, they are considered to be the first polymers for which PDA is a core component. It has become an attractive tool to extend the functionality of coated materials. al. reported that the PDA intermediate coating facilitates the physical adsorption of PEG and the stability of covalent bonding. It was demonstrated that the surface can be grafted qualitatively. 81 Proks et al. It was shown that G can be used as both an antifouling agent and a crosslinking agent. 82 They said PDA was coating Immobilization of amine-PEG-alkyne on a coated silicon wafer followed by azide functionalization After peptide immobilization, the surface exhibited improved binding of target cells. and maintained its repulsive properties against non-specific proteins. 82 Biologically active molecules and In combination, PEG modification has exciting potential for providing new bioactive materials. do.

[0211] Current techniques for antibody immobilization: Effective immobilization of antibodies onto surfaces is essential for biosensors, bioanalytical techniques, and biomedical devices. Potential to improve instrument development 83、84 . Non-covalent immobilization techniques include physical adsorption 85~91 Inert surfaces, either by encapsulation or by entrapment of antibodies within the coating matrix. These techniques are common methods for immobilizing antibodies on surfaces. Although successful, they can result in up to 90% antibody blockage due to blocking of the antigen-binding site. results in randomly oriented antibody molecules that remain inactive 92~94 Confinement method Using this method, we demonstrated biological activity in a pig model using dextran coating. This method creates a blend of dextran and the desired antibody. , then the dextran-antibody mixture is applied to the substrate using plasma reactor technology; This method creates a coating that exposes a fraction of the antigen-binding sites. demonstrated successful capture of the vesicles. 63 However, this coating can be used to Body (H-2K k When the surface was immobilized with IgG, the immunobinding activity was very poor. The presence of embedded antibodies was demonstrated on dextran / antibody coated SS discs. Although it was done, the surface is H-2K k It was not possible to capture expressing EPCs. The lack of effective cell capture may be due to non-oriented antibody immobilization (anti-H-2K embedded in dextran). k Antibody F This may be due to a combination of antibody denaturation (having many of the ab domains) and antibody degradation. When applied to FE graft materials, the dextran / anti-CD34 coating exhibited two distinct had no effect on the binding of circulating EPCs in a porcine AV shunt model 95、96 These The results show that dextran coating is effective in certain cases but not universally effective for antibody immobilization. This indicates that the ISA does not provide a viable method for

[0212] An alternative immobilization method involves non-specific target chemical immobilization (Figure 1). It utilizes a functionalized surface that reacts to exposed amino acid side chains. One limitation of this method is that it requires a The main problem is that we cannot control whether the antibody binds via the Fab or Fc side chains. Although specific functional groups are targeted, antibody orientation is still random. Similarly, this results in denaturation of the antibody and loss of immune binding activity. 97~100 .this The nonspecific technique by [End Page 111] et al. shows some promise, but, like the physical methods mentioned above, They are only effective in certain cases and no method is applicable to all surfaces. It is therefore applicable to many different surfaces and allows for the immobilization of antibodies in an oriented manner, It is important to develop new immobilization methods in which the b region is fully exposed and available for antigen binding. desirable.

[0213] Oriented antibody immobilization: As mentioned above, the immobilization process does not block binding sites or denature the antibodies. often resulting in partial or complete loss of immune binding capacity 101 To overcome this problem The technique involves immobilizing antibodies with their Fc domains immobilized and their antigen-binding Fab domains fully exposed. This involves immobilizing the material in an oriented manner. 102 Most antibodies have heavy chain Fc It is well established that the soluble carbohydrates in the soluble carbohydrate region of the soluble carbohydrates are at least one N-linked carbohydrate. Recently, an increasingly popular immobilization strategy involves modifying the oligosaccharides found in the Fc domain to bind to the antibody. It involves the introduction of novel reactive moieties into the antibody structure. There are two types of oligosaccharide modifications that have been used: the first is the addition of F to provide a reactive aldehyde group; accompanied by oxidation of oligosaccharides found in the c region 103、104 After oxidation, the newly formed The aldehyde moiety may be covalently attached to the amine-terminated surface. 105、106 Recently reported Another technique that has been proposed is to use a mutant β1,4 galactosyltransferase enzyme to convert the natural The acetylglucosamine residue is replaced with a modified sugar, which is a ketone or an azide. They often have unique chemical handles incorporated into their molecular structure. This introduces an Fc-specific target that can be used to immobilize the antibody. In this case, antibodies are coupled to cyclooctadecyl groups in an oriented manner via a catalyst-free "click" cycloaddition reaction. The antibody may be covalently bound to a surface having a specific Fc region. Therefore, both of these techniques provide covalent immobilization of antibodies with exposed Fab regions. (Figure 1).

[0214] Yuan et al. 107 A study by successfully applied anti-CD34 antibodies to SS slides. They demonstrated the effectiveness of oligosaccharide oxidation by selectively immobilizing α- and β-glucan as a cross-linking molecule. 3-Aminopropyltriethoxysilane was used to create an amine-rich surface and functionalize it with S S was immersed in an oxidized antibody solution. The oriented antibody retained its immunobinding ability and was not affected by conventional immobilization strategies ( demonstrated a three-fold increase in cell capture efficiency when compared to glutaraldehyde 107 Ka ng et al. 108 By immobilizing anti-mouse IgG antibodies on magnetic particles, This immobilization method was further investigated. Again, an amine-rich surface was created, and hydrazide copolymers were used. A coating was formed on the magnetic particles. Hydrazides react with aldehydes at lower pH. This has the advantage of preventing the formation of non-ionic surfactants between the amine residues on the antibody and the newly formed aldehyde. Prevent specific cross-linking. Orienting antibodies is accomplished by amine coupling (N-hydroxysuccinimide ) showed a two-fold improvement in immune binding efficiency compared to 108 .

[0215] Although still a relatively new technology, the enzymatic introduction of unique chemical moieties into the Fc region is also impressive. Boeggeman et al. used this technique to have functionalized several monoclonal antibodies (mAbs) with amines or fluorescent molecules. Streptococcus pneumoniae ia) β1,4 galactosidase to remove sugars found in the heavy chain region of the antibody; They then used a mutant β1,4 galactosidase to expose the terminal N-acetylglucosamine residue. Ketosilyltransferase (β1,4-Gal-T1-Y289L) enzyme was used The modified sugars were then introduced with an aminooxy group chemical handle. The results showed that the desired molecule (A Not only were the N-linked ATPs (lexa 488 and biotin) successfully incorporated into the antibody structure, but Conjugation of desired molecules to mAbs via conjugated carbohydrates modifies the affinity of the antibody for the antigen. showed that there was no 109 Zeglis et al. used a similar approach to radiolabeling β1,4 galactosidase B. Again, they first Glycosidase is used to remove the sugars found in the heavy chain region of the antibody, leaving terminal N-acetylglucosidase. They then used the same mutant enzyme (β1,4-Gal-T1-Y2 89L) was used to incorporate azide-modified sugars into the Fc region of the antibody. The modified antibody was then linked to a desferrioxamine-modified dibenzoate via a catalyst-free "click" coupling. Finally, the chelator-modified antibody was reacted with cyclooctyne. 89 Radiolabeled with Zr Their results were: 89 Zr successfully binds to the Fc region of the antibody and its incorporation into the antibody structure showed that the affinity of the antibody for the antigen was not affected by the 110 .

[0216] Study design: Polydopamine membrane: A PDA anchor will be used for the initial functionalization of all materials. l (dopamine hydrochloride, Sigma-Aldrich) solution (2 mg / ml) 10 mM The substrate (SS, CoCr, electrospun) is prepared in Tris-HCl (pH 8.5). The material (polyurethane thread, ePTFE graft material) was immersed in the solution for 24 hours in a dark environment. After the reaction, the material is removed, thoroughly rinsed, and dried under a stream of pure nitrogen. 111、112 . I They have used PDA membranes with 316L SS and CoCr discs, ePTFE graft materials, and other materials. These results were reported by Lee et al. t al. 71 Together with the results reported by

[20] , this functionalization method is feasible and This indicates that the PDA membrane can be applied to general cardiovascular platforms. Immobilization of biomolecules (BSA and avidin) on PDA-coated materials This has been demonstrated by the creation of various pre-bioactive surfaces through coating with 2% BSA solution. Simple immersion of the coated SS substrate created an antifouling surface that effectively inhibited cell adhesion. PDA-coated SS and COCR substrates exposed to avidin solution showed The capture of biotinylated fluorescent molecules was demonstrated.

[0217] Functionalization of polyethylene glycol: For the formation of a polyethylene glycol cross-linked layer, functionalized PEG amine (hydrazide or Dibenzocyclooctyne (DBCO) functionalized solution (25 mg / mL) was prepared in phosphate buffer solution. It will be prepared in physiological saline (PBS, pH 7.4). The pH of the solution will be adjusted to 8.6. The PDA coated material was immersed at 50°C for 30 hours. The material will then be thoroughly rinsed and dried under a stream of pure nitrogen. 113 We are The mono-amino PEG chains were successfully immobilized on the PDA-functionalized material. The surface was coated with PDA by immersing the substrate in a solution of amino-PEG4-DBCO. The DBCO surface was coated with azide-functionalized fluorescent molecules. This demonstrated effective capture of the offspring (Figure 9).

[0218] Biocompatibility assessment: ISO 10993 Guide to Biocompatibility Testing and Preclinical Evaluation of Biomedical Devices It will be done according to the line 114~117 , using 60 New Zealand white rabbits. The rabbits were treated with ketamine (40 mg / kg) and xylazine (5 mg / kg). The animal will be sedated with an intramuscular injection of 0.0 ... m 2 The skin is disinfected with isopropyl alcohol and A single incision approximately 8 cm long is made along the midline of the spine. The paraspinal muscles are then exposed. The muscles are incised approximately 1 cm parallel to the fiber axis, and a small pocket is created. Hemostasis is achieved by applying direct pressure. Pieces (5 x 5 mm) of coated test materials (SS, CoCr, electrospun polypropylene) ePTFE) and four uncoated controls were placed over at least 1 cm They will be implanted in a random fashion (four on each side) into the left and right paraspinal muscles. The flesh incision, subcutaneous tissue, and fascia are closed with absorbable sutures, and the skin incision is closed with non-absorbable sutures. The wound will be closed with sutures or skin staples. Animals will be monitored for 1, 7, 14, and 28 days. and sacrificed at 12 weeks by lethal intravenous injection of pentobarbital. Expose the implant site and observe signs of bleeding, necrosis, fluid accumulation, discoloration, infection, or encapsulation. Tissue from the implantation site will be collected and analyzed for symptoms. The tissues were fixed in marine. Final tissue analysis consisted of gross and histopathological data. We have extensive experience with this model and have used various stent platforms. foams and coatings, as well as ePTFE and bioabsorbable stent materials. It is used for this purpose.

[0219] Surface coating verification: The U.S. Food and Drug Administration (FDA) recently evaluated the safety and effectiveness of coated devices. provides guidance to the intravascular device industry for evaluating 118 Coating adhesion Characteristics such as solubility, barrier effect, and stability need to be evaluated.

[0220] Surface coating thickness: The thickness of vascular stent struts seen at follow-up It is well established that the degree of neoplastic hyperplasia is directly related to 119 Therefore, It is important to determine the thickness of the coating. S, CoCr, electrospun polyurethane, ePTFE) by contact profilometry (KLA Tenc or P16+, Surface Interface (SI) Ontario, Uni The analysis will be carried out using the University of Toronto. The coating is done so that only half of the surface is covered. The coated half and the coated The difference in height between the two halves of the profile is compared. All data will be managed by SI Ontario personnel using a secure protocol. The analysis will be conducted on-site under the guidance of SI Ontario staff. It will also provide information about the surface topography (roughness).

[0221] Surface coating uniformity, confluence, and barrier effectiveness: Scanning Electron Microscopy (SEM): Surface topography and integrity of coated surfaces The confluence and the size of the cells will be evaluated by SEM. (316L SS, CoCr, electrospun polyurethane, ePTFE) were subjected to standard dehydration and The nanotubes were prepared using a 20 nm gold sputter coating technique. A mirror (Philips XL-30 series, Netherlands) was used. become.

[0222] X-ray photoelectron spectroscopy (XPS): XPS is a method for determining the empirical formula, chemical state, and electronic state of elements on the surface of a material. This is a quantitative spectroscopic test that calculates the state of the material. The kinetic energy and number of electrons escaping from the sample are measured. K-Alpha XPS instrument equipped with a color Al Kα X-ray source (SI Ontario) (Thermo Scientific) 316L SS, CoCr, electrospun poly The X-ray source is S. Ontario personnel will operate using standard protocols (45° detection angle, (Kα radiation from a standard aluminum X-ray source operated at 300 W). Surface coating thickness, uniformity The uniformity, confluence, and completeness can be assessed by XPS. In the case of XPS, signals from metal compounds from the substrate will appear. It has been shown to be more sensitive than SEM for assessing coating integrity and consistency. R 120 .

[0223] Adhesion and cohesion of the surface coating after deformation: Depending on the stent design and material, Plastic deformation occurs in critical areas of the tent 121~126 According to finite element modeling , this plastic deformation is up to 25% 126 .

[0224] SEM: PDA coated surface (316L SS stent, CoCr stent) stent, electrospun polyurethane coated stent (PK-Papyrus stent, Bi otronik, Germany), ePTFE-covered stents (Jostent Graftmaster(TM), Abbott Vascular, Illinois The adhesion and deformability of the s) were evaluated by scanning electron microscopy before and after balloon expansion. Three of the devices will be attached to a standard angioplasty balloon for dilatation. Expanded stents were examined using SEM for evidence of cracking, flaking, or delamination. Luo et al. recently reported that PDA membranes were reported that vascular stents are resistant to deformation during compression and expansion. 127 . PDA membrane To assess the stability of the coating, the surface microstructure was examined for evidence of swelling or delamination. The results were analyzed using SEM before and after immersion in 37°C PBS for 7, 15, and 30 days. It will be an experiment.

[0225] XPS: 1cm diameter, 0.5mm thick coated 316L SS and Co The Cr disks were placed on a SATEC 3340 test system (Instron, Norwood, , MA, from OrbusNeich Medical Technologies, FL Plastic deformation of up to 25% was achieved using a punch test device attached to the All the variations are based on Lewis et al. 128、129 Described by To obtain a 25% deformation, a displacement speed of 0.05 mm / s and a maximum load of 2200 N were used. The adhesion and cohesion of the surface coating are measured using the above spectral analysis. This will be determined by analysis.

[0226] Surface hydrophilicity and surface energy: Water contact angle was measured by Kruss DSA machine and Drop Shape Analysis software (EasyDrop DSA20E, Kr by droplet analysis using a The droplet deposition and dispensing speeds were set to 5.0 μL and 195 μL, respectively, within 10 seconds of dispensing. The surface energy was measured by the water contact angle (polar solvent) and the diiodots. Using data for methyl ethane (a non-polar solvent), The calculation will be carried out by the method 130 .

[0227] Antibody coating: The ultimate goal of this proposal is to develop intravascular devices, such as coronary stents as a platform. As a proof of principle, we investigated the potential application of vascular grafts to localized drug delivery. E that have been genetically engineered to produce vasodilators (Section 4.1.2) for potential therapeutic purposes. The aim is to capture PCs that contribute to the endothelialization of the device through genetic engineering techniques. These genetically engineered EPCs can be used to generate EPCs that produce therapeutic compounds rather than just the EPCs themselves. A key issue with using PCs is that the instrument binds only and exclusively modified EPCs. Targeting CD34 ensures that rare modified cells and ubiquitous endogenous NDCs are isolated. To address this issue, we further engineered the cells to differentiate into two types of EPCs: By creating unique surface markers not naturally found on mouse cells, we can exclusively identify the compound-producing cells. Major histocompatibility complex class I molecules can provide targets for capture by the The H-2K k Some rare mouse strains (e.g., AKR / JA or CBA / J) ) and H-2K in other mammalian cells. k The absence of and H-2K kMonoclonal antibodies against surface proteins were used to exclusively capture engineered EPCs. pMACSKk.tag(C) plasmid vector makes it an attractive option for guaranteed acquisition. (Miltenyi Biotec) is H-2K k Cloning the gene and the gene of interest A bicistronic vector containing a multiple cloning site (MCS) that can be cloned Using the pMACSKk.tag(C) plasmid vector, we -2K k and the vasodilator calcitonin gene-related peptide (α-CGRP). The vector (pMACS-H-2K k -hCGRP, see also section 4.2.1) We built the H-2K k The antibody-coated vascular device is a genetically modified H -2K k It is believed that this will result in selective capture of expressing cells. "Eluting" stents produce minimal cell proliferation in the vessel wall only in the tissue adjacent to the stent struts. The goal of the described technique is to deliver a suppressive drug without any therapeutically relevant distal delivery. The goal is to deliver therapeutic amounts of bioactive compounds distal to the implanted device.

[0228] Oxidation method: t-Boc-hydrazide-PEG-amine (Quanta Biodesign) n) will be immobilized onto the PDA-coated material as described above (3.2. After successful immobilization of the PEG chains, the modified surface was treated with 25% trifluoromethane in methylene chloride. The sample was then subjected to trifluoroacetic acid (TFA) and rinsed with 10% ammonium hydroxide for 3 minutes. The t-Boc protecting group is then removed, creating a hydrazide-rich surface for additional immobilization. do 131、132 . anti-H-2Kk Antibody (IgG2a; Miltenyi Biote c, CA) to generate the required aldehyde moiety. 107、108 . anti The body is dissolved in PBS (0.05 mg / ml). Sodium (Sigma-Aldrich) was added to the antibody solution (metaperiod: 1 ml per ml of antibody). Sodium urate 2 mg), and the reaction is allowed to proceed in the dark for 30 minutes. After oxidation, the remaining Sodium metaperiodate was removed using a desalting column (Sephadex G-25). The PEG-functionalized material is then immersed in the oxidized antibody solution and allowed to react for 1 hour. Sodium cyanoborohydride (5.0M cyanoborohydride per 1 ml of antibody) Add 10 μL of sodium hydrazide to dissolve the hydrazide-rich coating. This stabilizes the Schiff base that is formed. The reaction is allowed to proceed overnight at 4°C. Finally, the material will be washed with PBS to remove physically adsorbed antibodies.

[0229] Enzymatic method: Amino-PEG4-DBCO (click chemistry tool) is coated on PDA. Anti-H-2K is immobilized on coated materials. k Antibodies were incubated according to the manufacturer's instructions. Click-IT® GlcNAc Enzymatic Labeling It can be modified using the ng System (Life Technologies). Briefly, the antibody (0.5 mg / mL in PBS) was applied to P30 resin (Bi Pre-treat using a microspin column prepared with o-Rad (1.5 mL total volume). The mixture is then buffer exchanged into a buffer solution (50 mM Na phosphate, pH 6.0). 0 μL anti-H-2Kk Add the antibody to the pretreated column and centrifuge at 850 x g for 5 minutes. The resulting antibody solution was added with 4 μL of β-1,4-galactosidase (Streptococcus aureus). Supplemented with S. pneumoniae (2mU / µL) and Place in an incubator at 37°C overnight. Tris-buffered saline (TBS, 20 mM Buffer exchange of samples into Tris HCl, 0.9% NaCl, pH 7.4 was performed using P30 This will be done using a microspin column prepared with resin. After buffer exchange 30 μL of antibody solution (2 mg / ml) was mixed with 4 μL of UDP-GalNAz (40 mM), 15 μL of MnCl2 (0.1 M), and 100 μL of Gal-T(Y289L) (0 0.29mg / mL) and incubated overnight at 30°C. After that, the antibody is buffer exchanged with PBS. Finally, DBCO is coated. The material was immersed in antibody solution (100 μg / ml) for 120 minutes and then washed with PBS. This will physically remove any attached antibodies.

[0230] Isolation of porcine endothelial progenitor cells: EPCs were obtained after culturing peripheral blood mononuclear cells in VEGF-containing medium. They are obtained in 4-7 days. They represent cells of hematopoietic origin and, through the release of paracrine factors, They exert angiogenic effects. They are classified as late EPCs (endothelial colony forming cells or late proliferating endothelial cells). Late EPCs (also called early EPCs) appear after 2-4 weeks of culture. Unlike early EPCs, late EPCs are They are thought to function as endothelial cells and can be incorporated into blood vessels. t al compared different cell sources and culture conditions and demonstrated bone marrow-derived EPs cultured for 3–4 weeks. We found that C is involved in the phenotype of endothelial cells. 133We adopted this method. The porcine EPCs used in the proposed study were procured using Ficoll-Polluted Porcine Bone Marrow Mononuclear Cells. Isolate by gradient centrifugation and place in fibronectin-coated T75 flasks 0.75×10 6 / cm 2 The cells were seeded at a density of 1000 and cultured in EGM-2 medium (Lonza). Our results show that porcine bone marrow-derived EPCs express two important endothelial markers, VE and VE- We have shown that these cells express GFR2 and eNOS. The EPCs used will be isolated from human peripheral blood mononuclear cells (PBMCs). These cells demonstrate similar properties to late EPCs. 133 .

[0231] Cell culture and genetic engineering: COS-1 cells and CHO cells (both from ATCC) ) supplemented with 10% FBS (Life Technologies) in Dulbecco's modified erythrocyte membranes. The cells will be maintained in Diagnosable Medium (DMEM, Life Technologies). Transfection of COS-1 cells was performed using Superfect® according to the manufacturer's instructions. Reagent (Qiagen), Lipofectamine Reagent for CHO cells This will be done using gent (Life Technologies). EPCs were nucleofected according to the manufacturer's instructions (Amaxa Nucleofector). We will transfect all modified cells using the cloning method. The cells are H-2K k It was shown that the surface protein is expressed.

[0232] Assessment of immune binding efficiency and cell proliferation: H-2K k Antibody-coated materials (31 6L SS, CoCr, electrospun polyurethane, and ePTFE) to H-2K k Expression cells In one embodiment, the antibody will be evaluated for its ability to selectively bind to the phospholipids. Polydopamine was directly immobilized onto the coated material in the absence of a carrier. Freshly prepared polydopamine-coated material was coated with anti-H2K in PBS. k antibody The coated material was then thoroughly rinsed with PBS, and the adsorbed The antibody was removed.

[0233] Detach the transfected CHO cells, wash them, and then incubate for 10 min. 6 PB at a density of cells / ml The antibody-coated discs and the coated discs are then resuspended in S. No discs, and discs coated only with the intermediate containing 2% BSA Block with PBS, incubate with 100 μl of cells, then completely wash with PBS. The bound cells were then washed thoroughly with 2% paraphoresis solution to remove unbound cells. The cells were fixed with methylaldehyde and nuclear stained with Sytox Green (Invitrogen). After that, the discs are visualized using a fluorescence microscope. A ctlaMax M5e plate reader (Molecular Devices) was used. Non-transfected cells will be used as a control. Our preliminary data suggest that immobilized H-2K k Antibodies detect H-2K on many different materials k This shows that the coating can capture CHO cells expressing IgG. To assess whether EPCs express H-2K kRemove the cells, wash them with PBS, and reintroduce them into the medium. The antibody-coated material (316 L SS, COCR, electrospun polyurethane, and ePTFE) (Each device was run in triplicate). At different time points after incubation (days 1, 3, and 5), samples were removed and resuspended in PBS. and washed with 0.25% trypsin-EDTA (Life Technologies). The adherent cells are lifted using the uncoated material. The number of cells in the culture medium will be compared with the number of cells in the culture medium.

[0234] Cell capture using an in vitro flow model: To test cell capture under flow conditions In vitro models of arterial blood flow have been developed in our laboratory. This model is available at Harvard Synthetic arterial device for stent deployment with flow controlled by a syringe pump The coated stent has a space therebetween, providing alternate flow through the vascular device. Uncoated stents and stents coated with only the intermediate 10 ml of transfected cells (10 5 cells / ml ) will be circulated at a flow rate of 3.1 mL / min for 1 hour. 134 Then, the stent is rotated. The cells are then harvested and washed with PBS to remove unbound cells. Cells will be fixed and visualized as described in section 3.2.5.5. The strength will also be measured as described in Section 3.2.5.5. Uninjected cells will be used for control purposes.

[0235] Our results demonstrate that anti-H-2K kH with antibody-coated vascular material -2K k 1 shows the capture of porcine EPCs expressing VEGF.

[0236] anti-H-2K k Cytotoxicity assay for antibody-coated ePTFE grafts The coated graft was then transferred to CHO H-2K k 1 with (+) cells, They were then fixed and imaged under a fluorescent microscope. Three sets were prepared each day. Additional spots on each graft were counted. One graft from day 1 was also fixed and taken for SEM.

[0237] Cell counts can be determined by imaging multiple focal planes and merging all the images together. Cells were considered to be ellipsoidal or spherical if they met the following parameters: (Size: over 12um x 12um and z: > 40um). This allows us to identify false positives. On day 1, the average The cell count was 960 ± 250 cells / mm 2 On day 2, the average cell count was 2595 ± 779 cells / mm 2 On day 3, the average cell count was 10,002 ± 1,745 cells / m m 2 (Figure 15).

[0238] Graft, CHO H-2K k (+) Incubated with cells for 1, 2, or 3 days They were then fixed (with multiple fixatives), critical point dried, sputtered with gold, and then SE. Multiple additional spots on each graft were analyzed. Only one graft was imaged. The number of cells increased, and the cell morphology changed from spherical to flat and polygonal. This occurs when cells grow and attach to the graft.

[0239] Research 2 We have used antibody-functionalized stents to deliver therapeutic substances using a novel cell-based delivery mechanism. We hypothesize that long-term intracoronary administration (local drug delivery) of

[0240] Background – Development of cell-based drug delivery systems: Vascular remodeling: The arterial wall is not a rigid tube, but rather remodels in response to hemodynamic, mechanical, and biochemical stimuli. Blood vessels expand to accommodate increased flow to downstream organs. has been known for over a century 135 A clear example of this process is The enlargement of coronary vessels during natural proliferation or myocardial hypertrophy. Radial expansion (outward or positive remodeling) is a progressive feature of atherosclerotic plaques tissue that can compensate for the proliferation of vasoconstrictors, thus delaying the development of flow-limiting stenoses stimulated by scientific observations 136、137 These pathological findings were subsequently The occurrence of outward remodeling in the presence of rheumatoid arthritis and how such outward remodeling occurs In vivo blood analysis revealed that plaque can hide significant amounts of plaque from angiographic detection. Supported by intraductal ultrasound (IVUS) studies 138、139 Most atheromatous The atherosclerotic segment shows some compensatory enlargement, but it does not completely maintain the lumen size. The effect is often insufficient to maintain normal blood flow, and some blood vessels may paradoxically shrink at the site of the lesion. (inward or negative remodeling), which may exacerbate rather than compensate for luminal loss. R 140 This type of constrictive remodeling occurs in 24% to 42% of coronary artery lesions. It has been reported that 141、142 The clinical significance of negative remodeling is Luminal narrowing is more closely related to the direction and magnitude of remodeling than to plaque size. This is emphasized by the observation that 140、143 .

[0241] In normal arteries, remodeling restores normal shear stress and wall tension, respectively. It is a homeostatic response to changes in flow and peripheral stretch to 144 Atheromatous Outward flow has been shown to occur in response to increased coronary flow from atherosclerotic monkeys. Remodeling 145 nitric oxide and matrix metalloproteinases (MMPs) It is highly dependent on fabricating a shear-responsive endothelium 146、147 Shear Sensitivity Remodeling Most of the mediators of stretching are also stretch-responsive, providing an important interface between stretch and shear signals. There appears to be an interaction 148 Vascular elasticity is a major determinant of resting vessel size. Recent data suggest that changes in elastin synthesis may also be important in remodeling. This suggests that 149 .

[0242] The presence of cardiac risk factors also influences the remodeling process. Remodeling and negative remodeling were more common in insulin-treated than in non-insulin-treated diabetic patients. Phosphate use is more common in smokers compared with nonsmokers in diabetic patients. 1 50、151Paradoxically, the frequency of negative remodeling is higher in hypercholesterolemic individuals. Low degree 152 Transplant failure is the most common cause of graft failure and death after heart transplantation. Vascular disease is characterized by diffuse angiographic narrowing and progressive intimal thickening. In addition, negative or insufficient positive remodeling occurs in transplanted hearts. It has been found to be common 152 .

[0243] Vasodilators: Prostacyclin: Prostacyclin (prostaglandin I2, PGI2) is A member of the prostaglandin family of lipid mediators and a potent vasodilator It has anticoagulant and antithrombotic effects. 153、154 Prostacyclin is a specific G-protein Protein-coupled receptors, IP receptors and / or nuclear receptors, peroxisome proliferators It is an autocrine and paracrine mediator that binds to activating receptor (PPAR) δ. be 155~158 Prostacyclin has local anticoagulant and vasodilator properties. It exerts a potent, non-storable, inactive metabolite, 6-keto prostaglandin F1α (PGF1α) Prostacyclin is rapidly converted to adenylate by a non-enzymatic process. induces relaxation of vascular smooth muscle via the inosinase / cyclic AMP transduction system and causes vasodilation in all vascular beds studied. 159 .

[0244] Stable prostacyclin analogs are clinically useful in the treatment of patients with peripheral and pulmonary vascular diseases. Although it is used continuously, its use is hampered by the fact that the substance is unstable and requires continuous administration. hindered by the fact that 154、160This limitation makes continuous administration of prostacyclin Preclinical investigations have been conducted into gene transfer techniques to provide delivery of human prostacyclin. Introduction of the PGIS gene has been shown to improve vascular diseases such as primary pulmonary hypertension. 161~16 3 and restenosis after vascular injury 164~166 To provide effective gene therapy for is shown.

[0245] Calcitonin gene-related peptide (α-CGRP): α-CGRP is a neurotransmitter that is secreted centrally and peripherally. It is distributed throughout the nervous system (vascular plexus) and exhibits biological effects, including effects on the cardiovascular system. GRP is one of the most potent arterial and venous vasodilators ever identified, and The potency of is approximately 10 times greater than that of prostaglandins and is comparable to that of other classical vasodilators (e.g., adenosine triphosphate). cetylcholine, adenosine, 5-hydroxytryptamine, and substance P) It is 0-1000 times more potent than the related peptide adrenomedullin. do.

[0246] There are several mechanisms by which α-CGRP induces vasorelaxation, mediated through the CGRP1 receptor. There are 167~169 Current evidence supports the role of both endothelium-independent and endothelium-dependent pathways of NO. Endothelium-independent mechanisms have been investigated in previous studies, including in porcine coronary arteries. observed in most tissues 170 α-CGRP stimulates these cells in the absence of endothelium. The ability to relax tissue is 172、173 It has been demonstrated in vitro, including It acts directly on SMCs to stimulate adenylate cyclase and intracellular cAMP production. These results suggest that α-CGRP inhibits voltage-dependent calcium release in smooth muscle cells for 1 hour. It stimulates 350% within 24-48 hours and increases the density of dihydropyridine receptors in the muscle cell membrane. has been shown to increase blood sugar by 30% 171 There is also an endothelium-dependent pathway, which induces NO secretion. Both cAMP and cGMP increase significantly depending on 174 CGRP inhibits endothelial-derived NO Due to its ability to stimulate vasodilation in the absence of vasopressin, it is characterized by reduced eNOS activation. This makes it an attractive agent for use in patients with endothelial dysfunction.

[0247] In many species and humans, coronary arteries are innervated by a high density of α-CGRP-containing nerve fibers. receiving 175、176 α-CGRP dilates coronary arteries at the site of atherosclerotic narrowing and may have a protective effect by delaying the onset of myocardial ischemia in patients with chronic angina. It is believed 177 Systemic administration of α-CGRP counteracts the deleterious effects of CAD and ischemia The therapeutic potential of α-CGR is limited by the effects of systemic administration, which leads to adverse effects. The most important aspect of P's activity is its potency as a peripheral vasodilator. The need for local administration of α-CGRP to achieve this effect is a key factor in target gene delivery. This means that it may be a relevant treatment.

[0248] Study design: Plasmid construction: Human α-CGRP complete cDNA (Open Biosystems, Huntsville) le AL) was used for PCR amplification of the DNA sequence encoding biologically active mature CGRP. The mature CGRP cDNA was then cloned into the vector pFLAG-CMV3 (Sigma). The FLAG epitope was fused to the FLAG epitope by inserting it into the HindIII / EocRV site of We created a clone expressing mature CGRP tagged with FLAG. This facilitates the identification of mature α-CGRP expression using anti-FLAG antibodies. The FLAG-α-CGRP cassette was then inserted into pMACSK k .tag(C) vector Inserted into the EcoRV / EcoRI sites of the double (H-2K k and α-CGRP) expression vector pMACS-H-2K k -Produces hCGRP. Prostacyclin synthase cDNA and pMACSK k The fragment will be cloned into the .tag(C) vector.

[0249] Recombinant lentiviral vectors: In addition to transfection with plasmid vectors (section 3.2.5.4), proof-of-principle For clarification, lentiviral vectors were used to transduce EPCs and induce long-term gene expression. The recombinant lentivirus will be provided by Cell Biolabs Inc. (San Diego, USA) will custom manufacture the device. We have successfully used lentiviral expression systems in previous projects to express EPCs and immortalized Extensive experience in lentiviral transduction of cell lines 178 .

[0250] Measurement of vasodilator expression: Measurement of α-CGRP expression and activity: α-CGRP expression was measured using anti-FLAG antibody (Sig This will be determined by Western blotting analysis using ELISA. Vector pMACS-H-2K k -hCGRP-transfected COS-1 cells showed that conditioned medium (CM) contains CGRP. The biological activity of CGRP was confirmed in humans. As assessed by its ability to induce nerve growth factor (NGF) production in keratinocytes. R 179 .

[0251] Measurement of prostacyclin synthase expression and activity in transfected cells The prostacyclin synthase that is involved is an antibody against human prostacyclin synthase. Determined by Western blotting using (R&D Systems) Prostacyclin synthase activity was measured by radioimmunoassay according to the manufacturer's instructions. The metabolite 6-keto-PGF1α in CM was measured using a ELISA (Amersham Corp). The evaluation will be carried out by determining the

[0252] Transgenic expression timeline: We used pMACS-H-2K k -hCGRP CHO cells transfected with α-H-2K for up to 5 days k While producing proteins, We demonstrated that cell morphology and viability were unaffected (data not shown). On days 1, 3, 5, and 7 after the infection, the plasmid-transfected EPCs and lentivirus were transfected. Stability of gene expression (H-2K) was observed in both transduced and non-transduced EPCs. k and vasodilators ) will be determined.

[0253] In vivo cell capture: All experiments will be performed in young male Yorkshire pigs (>30 kg). Prior to device implantation, hyperemia was observed in the coronary arteries. Administration of 200 μg of glycerin induces coronary artery atherosclerosis in the three major coronary arteries. Arterial angiograms will be obtained and online quantitative coronary angiography (QCA) will be performed. The coronary artery cross-sectional area (CSA) of the vessel segment distal to the implantation site was measured by intravascular ultrasound (IV). Doppler-derived blood pressure will be determined by ultrasound (US) and optical coherence tomography (OCT). Flow velocity was measured using a 0.014-inch steerable Doppler guidewire (ComboWire) XT, Volcano Corp., San Diego, CA) and C The mean coronary peak flow was analyzed using the ombomap system (Volcano Corp.). Volumetric coronary blood flow (CBF) will be reported as a previously validated It is calculated using the relationship CBF=CSA x APV. 180 Stent Platform For evaluation of cell capture in the form, an 8 mm long COCR coronary stent was placed in the PDA / P EG / anti-H-2K k The stent was coated with 1.1:1 stent-to-vessel ratio and three main The catheter will be randomly deployed in the proximal segment of the important epicardial coronary artery. To assess cell capture, PDA / PEG / anti-H-2K k Coated Jos Tent Graftmaster Coronary Stent Graft (between two SS stents) Sandwiched ePTFE (Abbott Vascular) will then be used. Cell administration was performed using a prototype tandem balloon catheter (kindly provided by Cordis C The catheter will be used to It consists of two tipped, highly compliant balloons that are inflated through a single inflation port. Once inflated, a 1.0 cm long local infusion chamber is created between the balloons. Distal blood flow is provided by the central lumen, and solutions are delivered to the chamber through two separate ports. The tandem balloon can be inflated or suctioned. When inflated to 100°C, saline is pumped through the infusion port to clear the blood chamber. The central arterial segment was then k and prostacyclin or α-CGRP or Sky Vector (H-2K k 3× 10 6 Randomization will be required to receive the EPC. k+ Before delivery of EPC Follow the manufacturer's instructions to use the MACSelect Kk System (Miltenyi The cell suspension was concentrated using a centrifuge tube (Biotec). 2 ml of the cell suspension was then concentrated at 200 μL / min. The infusion rate will be administered over 10 minutes, followed by a 10 minute dwell time. The incision site is closed and the animal is allowed to recover. For both implanted EPCs, two stents (16 protacyclines) were implanted per animal. α-CGRP synthase (8 COCR, 8 ePTFE) and 16 α-CGRP (8 A total of 64 animals were treated with COCR, 8 ePTFE, and their respective controls. Two animals from each group were to be sacrificed 5 days after stent implantation. Coronary angiography and QCA will be performed to identify the stented segment. The detached arterial segment is then bisected longitudinally, and one half is then inserted into a standard Analyze by histochemistry and process one half for SEM imaging. The segments for histochemical analysis were placed in 10% formalin / PBS solution and separated into five sections. Sections were cut and stained with hematoxylin and eosin (HE) and elastin trichrome. The degree of neoplastic hyperplasia and the inflammatory score (Kornowski A score (0-3) will be determined to assess evidence of rejection of the delivered cells. R 181 The segments were fixed in 10% buffered formalin / PBS for 30 seconds. The sections were then prepared for SEM and further soaked in 0.1 M sodium cacodylate buffer (Sigma) overnight. ) in 2% PFA with 2.5% glutaraldehyde (BDH Inc.) Post-fixation was performed with 1% osmium tetroxide (S) in 0.1 M cacodylate buffer. This is completed by dehydration with ethanol, followed by critical point drying. The samples were then gold sputtered at the University of Toronto SEM facility according to established protocols. SEM will be performed to assess surface endothelialization. Twenty-eight days after the index procedure, the remaining animals (6 per group) were anesthetized and Q Coronary angiography with CA analysis will be performed. The vessels will then be analyzed using IVUS and OCT. The coronary artery Doppler flow will be measured and CBF will be calculated. We found that in animals receiving vasodilators that express EPCs, the stented segment We expect a significant increase in vascular diameter beyond that of the previous study.

[0254] Expected Results: Antibody functionalized materials: We demonstrated that PDA / PEG surface modification provides an effective platform for antibody immobilization. demonstrate that it can be used to create bioactive coatings on a variety of biomedical materials This technology is expected to be useful for the development of pro-healing devices and for the application to target tissues in vivo. It has application as a platform for the local delivery of therapeutic compounds.

[0255] Potential confounding factors are the unpredictability of antibody modification, insufficient immobilization and denaturation of the antibody, The oxidative and enzymatic techniques described may result in the production of several of the same isotypes. This effectively immobilizes the antibody to the substrate, but the sugar moieties on the antibody are glycosylated and difficult to access. The degree of roughness is variable 182 The immobilization techniques described provide sufficient binding for in vivo applications. If this is not possible, alternative fixation strategies will be explored. Of particular interest are new It is a UV fixation technique that utilizes indole-3-butyric acid-PEG to A conserved nucleotide binding site found on virtually all antibodies, regardless of type Binding of antibodies via 183 .

[0256] Cell-based drug delivery systems: We demonstrate that this unique cell-based intracoronary administration of a potent vasodilator enhances the positive response of porcine coronary arteries. We hope that this will promote Tibli modeling. This could translate into a viable clinical treatment for patients with "no options" for revascularization. Proof of principle of the technique is provided. Possible clinical benefits include positive remodeling of conduit coronary arteries. flow-mediated arterial flow not only from the coronary artery but also from the ischemic area not supplied by the feeder coronary artery This technology allows the delivery of numerous therapeutic compounds to various target tissues within the body. It may also be used for delivery.

[0257] H-2K k The antigenic load of surface proteins is very low and is unlikely to induce a cellular immune response. Although unlikely, there is evidence of cellular wasting or inflammation in the vessel wall early after stent implantation. If there is a surface marker / antibody system, we will use H2Kk / anti-H-2K k from ΔLNGFR / Anti-LNGFR (Miltenyi Biotec, CA) will be used. There is increasing evidence that the NGFR surface marker is poorly immunogenic. 184 , myocardial infarction pigs Evidence for long-term survival of autologous mesenchymal stem cells expressing ΔLNGFR in a model has been published. It is being 185 .

[0258] Example 4: Polydopamine-PEG-antibody stent coating Polydopamine (PDA) coating Stents (stainless steel and CoCr) were sonicated for 5 minutes in the following solutions: desorption Ionized water, acetone, ethanol, and water. The stents were then dried under air. The bovine pericardial graft was not sonicated to avoid protein denaturation, but excessive lysis was prevented. To remove the free aldehyde, the grafts were incubated in PBS (pH 7.2) for 24 hours, then Prewash with 0.5 M TRIS-HCl (pH 6) for 1 hour, then rinse with deionized water. Furthermore, the pericardium and ePTFE graft were not air-dried, but were diluted with water to 10 mM Tris The solvent was exchanged into s-HCl buffer (pH 8.6). The stent was then placed in an orbital mixer. The solution was incubated at room temperature for 24 hours in 10 mM Tris-HCl buffer (pH 8.6). Polydopamine was prepared by dip-coating in a polydopamine solution (2 mg / ml). The ePTFE and bovine pericardial grafts were coated (Fig. 4A-B). For the test, a solution of 5-10 mg / ml dopamine hydrochloride was used.

[0259] PEG coating 25mg / ml t-Boc-hydrazide in Tris-HCl buffer (pH 8.6) -PEG8-amine MW: 555.66 g / mol (Quanta Biodesign The PDA-coated stents were rinsed with deionized water before reaction with HCl. The procedure was carried out at 50°C for 24 hours (Figures 6 and 7). The stents were then washed at room temperature. ePTFE and bovine pericardium were washed with water, acetone, and dichloromethane without drying. Note that a solvent exchange to dichloromethane (DCM) was performed. The PEGylated stents were then in DCM for 5 h at room temperature with constant airflow and venting to release CO by-product. Deprotection was performed by removing the t-boc functional group with 2 mg / ml iodine (I2). Once the reaction was complete, the stents were then washed with DCM and dried under nitrogen / argon. For bovine pericardium and ePTFE grafts, the endothelial cells were removed from the DCM prior to further processing. The material was kept wet by solvent exchange to ethanol and deionized water.

[0260] Antibody oxidation The selected antibodies were incubated in a buffer containing 20 mM sodium acetate and 15 mM sodium chloride. The antibody was dissolved in buffer at a pH of 4 to 6.5. The antibody was then oxidized with sodium periodate. The reaction flask was covered with aluminum foil to prevent exposure to light (Figure 5). A PD Mini Trap G-25 column (GE Life Sciences) was used. The purified antibody was purified by column chromatography using a 100% ethanol solution. This was confirmed by optical spectroscopy.

[0261] Antibody conjugation to PDA-PEG The PDA-PEG coated stent was immersed in a purified antibody solution. The tents were removed, washed, and placed in PBS pH 7.2 until further testing (Figure 6 and Figure 7).

[0262] Example 5: Coating biocompatibility: cytotoxicity assay The cytotoxicity of anti-CD34 antibody-coated ePTFE surfaces was evaluated. C (approximately 30% CD34 positive) were seeded on the coated surface. 24 hours after seeding and After 48 hours, cells grown on the surface were stained with a fluorescent dye and observed under a fluorescence microscope.

[0263] [Example 6] The aim of this study was to improve distal flow using a technique of localized capture of genetically modified EPCs. To improve coronary remodeling, we aim to develop novel methods to promote positive coronary remodeling. We have demonstrated that chronic intracoronary administration of potent vasodilators increases coronary blood flow and induces vascular We hypothesize that positive remodeling of the arteries increases the vessel diameter. Targeted Delivery of EPCs Genetically Modified to Express a Flow-Dependent Agent to Epicardial Coronary Arteries The aim is to promote positive sexual remodeling.

[0264] PDA / PEG / anti-H-2K k Antibody-coated stents (e.g., cobalt chromium stents, 9 mm long) in vitro or in subjects (e.g., experimental animals or patients) Implanted upstream of the target vascular site.

[0265] Genetically engineered cells (e.g., bicistronic genes encoding PGIS or α-CGRP) vector) into an in vitro system or a subject, e.g., a standard It is delivered through a wire port on a balloon catheter and released into the isolated lumen of the blood vessel. The engineered cells proliferate and express proteins such as PGIS and α-CGRP (cells are shown in Figure 1). It is predicted that CGRP expression will continue for more than 6 days (Nagaya et al. 200 3) The α-CGRP protein travels downstream and attaches to the CGRP1 receptor. The blood vessels expand in response to the released GGRP protein. Long-term effects include positive changes in blood vessels. Includes fibre modeling.

[0266] method Genetic modification of EPCs Isolate porcine bone marrow-derived EPCs and culture them according to a previously established protocol in our laboratory. The double gene (H-2K k and human α-CGRP) expression vectors were constructed and electroporated. For example, truncated mouse MHC class B12 was introduced into EPCs using troporation. I molecule H-2K k Plasmid pMACS K expressing k .II(Miltyneyi Genetically modified EPCs were analyzed by flow cytometry and immunohistochemistry, respectively. Western blotting revealed H-2K k and assayed for α-CGRP production α-CGRP bioactivity was assayed.

[0267] In vitro cell binding assay anti-H-2K k Block the antibody-coated substrate with 2% BSA in PBS did.

[0268] H-2K k H-2K expression in EPCs was blocked with BSA. k Ab coated The unbound cells were mixed with the substrate and incubated at room temperature for 1 hour. The bound cells were washed away, fixed, stained with the fluorescent nuclear dye Sytox Green, and visualized under a fluorescent microscope. The cells were observed under a microscope, and the fluorescence intensity was also measured using a fluorescence reader.

[0269] result Dual expression of H-2K by genetically modified porcine EPCs k and α-CGRP) 67% of porcine EPCs expressed H-2K 24 hours after gene modification k Dual expression vector The vector-modified porcine EPCs also express α-CGRP.

[0270] α-CGRP bioactivity assay CGRP bioactivity upregulates nerve growth factor (NGF) expression in keratinocytes The antibodies were assayed for their ability to bind to the antibody, for example, in an ELISA assay.

[0271] conclusion New coating technology is available for a variety of materials (stainless steel, cobalt chrome, ePTFE) , pericardium, etc.

[0272] Anti-CD34 antibody and anti-H-2K k Antibodies such as antibodies can be produced by new coating technology. It can be immobilized on a variety of vascular devices.

[0273] Porcine EPCs express the potent vasodilator α-CGRP and / or exogenous α-CGRP, which can be used for cell capture. Antigens such as H-2K k (or Cutaway H-2K k ) genetically engineered to express It is possible.

[0274] anti-H-2K k The antibody-coated vascular material exhibited H- 2K k (or Cutaway H-2K k ) can be captured.

[0275] [Example 7] The in situ accessibility of the Fab and Fc domains of immobilized antibodies was assessed using Sah Analyst 142:4247-4256 (2017) Fab domain accessibility assay - known amount of monoclonal Antibody, e.g., anti-CD34 coated device (e.g., disk, ePTFE glass) rafts, stents) to bind to antigens capable of binding to bound antibodies, e.g., soluble CD34 Incubation will be in molar excess (relative to the molar amount of bound antibody). This will saturate the available antibody domains. Wash the coated device and use a antibody that binds to an epitope different from the primary monoclonal antibody. Secondary 125I-radiolabeled monoclonal antibody (relative to the amount of bound monoclonal antibody) The device is incubated for a period ranging from about 1 hour to about 3 hours. The cells were washed with, for example, phosphate buffered saline (PBS), and the radioactivity (cps) was measured by gamma counter. The different pre-existing concentrations of 125I-radiolabelled secondary monoclonal antibodies in solution are measured. The stock of known concentrations will serve as a control. The amount of bound secondary radiolabeled monoclonal antibody in (a) was measured by the secondary radiolabeled monoclonal antibody. The final signal after binding of the monoclonal antibody was compared with the signal of the monoclonal antibody-coated device. The calculation is made by subtracting the signal. st 142:4247-4256(2017). Activity and accessibility of immobilized antibodies Other techniques used to determine orientation include atomic force microscopy, neutron reflectometry, Spectroscopic ellipsometry, and mass spectrometry.

[0276] Polyimide, such as grafts, stents, discs, nanoparticles (e.g., metal or polymeric), Dopamine-PEG-antibody coated substrates are prepared as described in Example 4. The antibody, e.g., a monoclonal anti-CD34 antibody, is then added to the polydopamine- The secondary anti-CD34 monoclonal antibody binds to the polydopamine-PEG moiety. The epitope on the CD34 molecule is different from that of the monoclonal anti-CD34 antibody bound to PEG. The secondary anti-CD34 monoclonal antibody directed against the target antibody will be radiolabeled (iodine). Iodination reagent (or "Iodo-gen"): 1,3,4,6-tetrachloro-3α,6α -diphenylglycouril), Thermo Fisher Scientific ( In some embodiments, the antigen comprises multiple epitopes that are the same. If the antibody has a target site, a second monoclonal antibody can be directed to the same site. Radiolabeled anti-CD34 monoclonal antibody against CD34 conjugated to 4-polydopamine-PEG Binding of the antibody to the antibody will be measured as follows.

[0277] Anti-CD34-polydopamine-PEG coated discs were placed in a PBS bath. Incubate for 1 hour with excess soluble CD34 (10 mM phosphate-buffered saline) saline, pH 7.4). A molar excess of CD34 was added to saturate the available antibody domains. After incubation, anti-CD34-polydopamine-PEG was The coated discs were washed twice with PBS buffer and then 125I radiolabeled dimers were added. Next, anti-CD34 monoclonal antibody was added for sandwich assay binding. A molar excess of the secondary monoclonal antibody will be used. After the injection, the anti-CD34-polydopamine-PEG coated discs were washed with PBS. Wash three times and measure the final radioactivity (cps) in a gamma counter using a final 100 μL plate. The cells will be resuspended in 1 L of PBS buffer. The amount of the antibody was determined by the signal after binding of a radiolabeled anti-CD34 monoclonal antibody. This will be the case.

[0278] Radiolabeled antibody onto polydopamine-PEG-antibody coated disks Binding of CD34 antibodies was confirmed using radiolabeled anti-CD34 antibodies lacking PEG or polydopamine. The bond between the coated discs is larger than that between the coated discs. is expressed as a mass ratio and a number ratio.

[0279] Cell adhesion can be assessed using a suitable method, such as a cell adhesion assay. Adherent cells can be quantified using colorimetric or fluorescent detection.

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Citation and discussion of such references is solely for the purpose of clarifying the description of the present invention. Any references are provided for the purpose of illustration only and are not intended to be a guarantee of prior art to the inventions described herein. All references cited and discussed herein are to be construed as an admission of art. The references herein are incorporated by reference in their entirety. Variations, modifications, and other implementations may occur to those skilled in the art without departing from the spirit and scope of the invention. While particular embodiments of the present invention have been shown and described, it is to be understood that the spirit and scope of the present invention are not to be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that changes and modifications can be made without departing from the scope of the present invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of example only and not by way of limitation. It is only available as is.

Claims

1. 1. A medical device having a coating, comprising: the medical device is a prosthetic heart valve, a vascular stent graft, or a synthetic graft; the coating comprises (i) polydopamine, (ii) a polyether derivative, and (iii) an antibody, wherein the polydopamine is coated on the medical device and covalently bound to the polyether derivative, and the polyether derivative is covalently bound to an Fc region of the antibody; the polyether derivative is a polymer having a polyether backbone, and the polyether backbone is polyethylene glycol (PEG), a polyethylene glycol (PEG) derivative, polypropylene glycol (PPG), a polypropylene glycol (PPG) derivative, or a combination thereof; Medical equipment.

2. The medical device of claim 1 , wherein the antibody specifically binds to a cell surface antigen of an endothelial progenitor cell or an endothelial cell.

3. The medical device of claim 2 , wherein the cell surface antigen is CD34.

4. The medical device of claim 1 , wherein the polyether backbone is PEG.

5. 10. The medical device of claim 1, wherein the polyether derivative has an average molecular weight in the range of 200 Daltons to 1000 Daltons.

6. 10. The medical device of claim 1, wherein the polyether derivative has an average molecular weight in the range of 200 to 350 daltons.

7. 10. The medical device of claim 1, further comprising a biocompatible polymer selected from the group consisting of polytetrafluoroethylene (PTFE), expanded polytetrafluoroethylene (ePTFE), polyethylene terephthalate, polyethylene, polyurethane, polypropylene, or combinations or derivatives thereof.

8. 10. The medical device of claim 1, wherein the coating further comprises a pharmaceutical agent.

9. 9. The medical device of claim 8, wherein the pharmaceutical agent inhibits smooth muscle cell migration and / or proliferation.

10. 9. The medical device of claim 8, wherein the pharmaceutical agent is paclitaxel, rapamycin, sirolimus, everolimus, tacrolimus, biolimus, biolimus A-9, or a combination thereof.

11. The medical device of claim 1 , wherein the antibody is monoclonal or polyclonal.

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