Coating which promotes endothelial cell adherence

The present invention relates to the field of medical devices that are implanted in blood vessels in the body. More specifically, the invention relates to a stent or synthetic implant that is implanted in a blood vessel that incorporates a matrix that facilitates adhesion of endothelial cells to the stent or synthetic implant.

This application claims the interests of US Provisional Application No. 60 / 189,674, filed March 15, 2000, and US Provisional Application No. 60 / 201,789, filed May 4, 2000.

Atherosclerosis is one of the leading causes of death and disability in the world. Atherosclerosis is associated with the deposition of fatty plaques on the luminal surface of the artery. The deposition of fat plaques on the luminal surface of the artery causes a narrowing of the cross-sectional area of the artery. Ultimately, this deposit blocks blood flow distal to the lesion, causing ischemic damage to the tissue supplied by the arteries.

The coronary arteries supply blood to the heart. Coronary atherosclerosis (CAD) is the most common and serious chronic life-threatening disease in the United States, affecting more than 11 million people. The social and economic costs of coronary atherosclerosis far exceed the costs of most other diseases. Narrowing of the coronary lumen causes destruction of the myocardium, first resulting in angina, followed by myocardial infarction and ultimately death. In the United States, more than 15 million people have myocardial infarction each year. Sixty-thousand (or 40%) of such patients suffer from acute myocardial infarction, and more than 300,000 of such patients die before reaching the hospital (Harrison's Principles). of Internal Medicine, 14th Edition, 1998).

CAD can be treated with percutaneous transluminal coronary balloon angioplasty (PTCA). Over 400,000 PTCA procedures are performed annually in the United States. In PTCA, a balloon catheter is inserted into a peripheral artery and passed through the arterial system into a blocked coronary artery. The balloon is then inflated, stretching the arteries and flattening the occluded fat plaque, thereby increasing the cross-sectional flow of blood through the affected arteries. However, this therapy usually does not result in a permanent opening of the affected coronary artery. About 50% of patients treated with PTCA require repeated treatment within 6 months to cure restenosis of the coronary arteries. Medically, this re-stenosis of the artery after treatment with PTCA is called restenosis. Restenosis is severely associated with vascular recoil and contraction. Subsequently, after vascular recoil and contraction, intermediate smooth muscle cells proliferate in response to PTCA damage to the artery. Somewhat smooth muscle cell proliferation is thromboxane A_２Is mediated by the release of various inflammatory factors from the injured area, including platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). The problem of restenosis has been overcome using a number of different techniques, including treatment of patients with mechanical retention of arterial openings with various drugs or stents (Harrison's Principles of International Medicine, 14th). Edition, 1998).

Of the various techniques used to overcome restenosis, stents have proven to be the most effective. A stent is a metal scaffold located in a diseased vascular segment to create a normal vascular lumen. Stent placement in the affected arterial area prevents arterial recoil and subsequent closure. Stents can also prevent local dissociation of arteries along the middle layer of arteries. By retaining a lumen larger than the one created using PTCA alone, the stent reduces restenosis by as much as 30%. Despite their success, stents have not completely ruled out restenosis (Suryapranata et al 1998. Random use of coronary stents with balloon angioplasty in selected patients with acute myocardial infarction. Comparison (Randomized comparison of coronary stenting with balloon angioplasty in selected patients with acte myocardial initiation). 97: 2502-2502).

Arterial stenosis can occur in non-coronary vessels, including the aortic circumflex artery, subclavian artery, deep artery of the thigh, distal patellar artery, tibial artery, subclavian artery, and mesenteric artery. The prevalence of peripheral atherosclerosis disease (PAD) depends on the specific anatomical site affected and the diagnostic criteria used to diagnose occlusion. Traditionally, doctors have used intermittent claudication tests to determine if PAD is present. However, this measurement can very underestimate the actual occurrence of the disease in the population. The proportion of PAD appears to change with age, and the incidence of PAD increases in the elderly. National Hospital Discharge Survey Data from Survey) show that 55,000 men and 44,000 women are newly diagnosed with chronic PAD each year, with 60,000 men and 50,000 women acutely diagnosed. It is estimated that a new diagnosis of PAD has been made. 91% of cases of acute PAD involved the lower extremities. The prevalence of coexisting CAD in patients with PAD can exceed 50%. In addition, there is an increased prevalence of cerebrovascular disease among patients with PAD.

PAD can be treated with percutaneous transluminal balloon angioplasty (PTA). The use of stents in combination with PTA reduces the occurrence of restenosis. However, postoperative results obtained using medical devices such as stents are not comparable to those obtained using standard surgical revascularization procedures, ie those with venous or prosthesis bypass materials (Principles). of Surgery, Schwartz et al. Eds, Chapter 20, Arterial Disease, 7th Edition, McGraw-Hill Health Services Division, New York 1999).

Preferably, the PAD is treated using a bypass method in which the arterial blocking surface is bypassed with a graft (Principles of Therapy, Schwartz et al. Eds, Chapter 20, Arterial Disease, 7th Edition, McGraw-Hill). Health Services Division, New York 1999). The implant can be composed of autologous veins such as saphenous veins or synthetic implants such as those made from polyester, polytetrafluoroethylene (PTFE) or foamed polytetrafluoroethylene (ePTFE). The rate of postoperative opening depends on a number of different factors, including the luminal size of the bypass graft, the type of synthetic material used in the graft, and the site of outflow. However, restenosis and thrombosis remain important problems even with bypass grafts. For example, the patency of the submandibular bypass method using the ePTFE bypass graft in the third year is 54% for the femoral-popliteal bypass and only 12% for the femoral-tibial bypass.

Therefore, there is often a need to improve the performance of both stents and synthetic bypass grafts to further reduce CAD and PAD morbidity and mortality.

When using stents, the approach was to coat the stent with various antithrombotic or restenotic agents to reduce thrombosis and restenosis. For example, impregnating a stent with radioactive material appears to suppress restenosis by suppressing the migration and proliferation of myofibroblasts (US Pat. Nos. 5,059,166, 5,199,939). No. 5 and 302,168). Irradiation of treated blood vessels can cause safety issues for physicians and patients. Moreover, irradiation does not allow uniform treatment of diseased blood vessels.

Alternatively, the stent is also coated with a chemical agent such as heparin or phosphorylcholine, both of which appear to reduce thrombi and restenosis. Heparin and phosphorylcholine appear to significantly reduce restenosis in short-term animal models, but treatment with these agents does not appear to have long-term effects on the prevention of restenosis. In addition, heparin can induce thrombocytopenia and cause serious thromboembolic complications such as seizures. Nevertheless, it is not feasible to load the stent with a therapeutically effective amount of heparin or phosphorylcholine in this manner to allow the treatment of restenosis to be practiced.

Synthetic grafts have been treated in a variety of ways to reduce postoperative restenosis and thrombi (Bos et al. 1988, "Small-Dimensional Vascular Graft: Current Status" (Small-Diameter Vascular Graft). Processes: Current Status) Archives Physio. Biochem 106.100-115). For example, polyurethane composites such as mesh polycarbonate urethane have been reported to reduce restenosis as compared to ePTFE implants. The surface of the implant has also been modified with high frequency glow discharge to add polyterephthalate to the ePTFE implant. Synthetic implants have also been impregnated with biomolecules such as collagen. However, none of these approaches significantly reduced the incidence of thrombi or restenosis over time.

Since endothelial cells have certain unique properties, such as cytoregulatory molecules that reduce the occurrence of thrombi or restenosis, stimulating the development of a single layer of endothelial cells on the surface of a stent or synthetic implant is re-stimulating. May prevent both stenosis and thrombosis (Belle et al. 1997. Stent Endotheliumization. Circulation 95: 438-448; Bos et al., 1998. Small-Diameter Endothelium Brazil -115).

Endothelial cells have been deposited on the surface of the stent by local delivery of vascular endothelial growth factor (VEGF), which is an endothelial cell mitogen, after stent transplantation (Belle et al. 1997. Stent Endothelialization. Circulation 95.438-448). This form of treatment may be unreliable, as the application of VEGF can have systemic as well as local effects.

Synthetic grafts have also been seeded with endothelial cells, but clinical results using endothelial seeding are generally poor, i.e., postoperative opening rates were low (Lio et al. 1998. New percentages and Materials in). Microvasural Grafting: Prosthetic Graft Endothelial Cell Seeding and Gene Therapy. Microsurgery 18: 263-256).

Therefore, there is a need to develop new methods and compositions for coating medical devices, including stents and synthetic implants, with endothelial cells. This type of coating will prevent not only restenosis, but also thromboembolic complications due to stenting. Methods and compositions that provide such improvements eliminate the disadvantages of prior art and have a significant positive impact on CAD and PAD-related morbidity and mortality. It is an object of the present invention to prepare stents and synthetic implants coated in a manner that stimulates adhesion of endothelial cells to medical devices such as stents or synthetic implants.

<p num="0018">The present invention provides methods and compositions for coating a medical device with a matrix that promotes adhesion of endothelial cells to the medical device. This matrix incorporates antibodies that stimulate the adhesion of endothelial cells to the surface of medical devices.</p><p num="0019">As used herein, "medical device" refers to a device that is temporarily or permanently introduced into a mammal for the prevention and treatment of medical conditions. These devices include any that is subcutaneously, transdermally, or surgically introduced and left in an organ, tissue, or lumen. Medical devices include stents, coated stents such as those coated with polytetrafluoroethylene (PTFE) or foamed polytetrafluoroethylene (ePTFE), synthetic implants, artificial heart valves, artificial hearts and artificial organs for vascular circulation. Fixtures for connection, venous valves, abdominal aortic aneurysm (AAA) implants, inferior aortic filters, permanent drug infusion catheters, embolic coils, embolic materials used for vascular embolization (eg, PVA foam) , And vascular sutures.</p><p num="0020">Coating of medical devices using the compositions and methods of the invention stimulates the development of endothelial cell layers on the surface of medical devices, thereby causing restenosis and other thrombi resulting from transplantation of medical devices. Can prevent embolic complications.</p><p num="0021">Synthetic implants and stents can be used to treat CAD or PAD. The stent or synthetic implant may be coated with a matrix that incorporates antibodies that stimulate the adhesion of circulating primordial endothelial cells to medical devices. The antibody may include a monoclonal antibody that is reactive with an endothelial cell surface antigen, such as CD34, which is an antibody expressed on the surface of primordial endothelial cells. Fab fragments of monoclonal antibodies may be used. In another embodiment, monoclonal antibodies against other endothelial surface antigens such as KDR or Tie-2 can also be used. In one embodiment, a single type antibody that reacts with one antigen may be used. Alternatively, a plurality of different antibodies against different endothelial cell surface antigens may be mixed together and added to the matrix.</p><p num="0022">The matrix that coats the medical device may be composed of synthetic materials such as polyurethane, poly L-lactic acid, cellulose esters or polyethylene glycol. In another embodiment, the matrix is composed of natural materials such as collagen, fibrin, elastin or amorphous carbon. The matrix may include multiple layers having a first layer composed of synthetic or natural materials and a second layer composed of antibodies. The layers are sequentially aligned, with one surface in which the first layer is in direct contact with the surface of the stent or synthetic implant and the second layer in contact with the first layer and the opposite surface in contact with the vascular lumen. May be done.</p><p num="0023">In a third embodiment, the matrix may comprise fullerenes, where fullerenes range from about C60 to about C100. Fullerenes may also be arranged as nanotubes, which incorporate molecules or proteins. The fullerene matrix may also be mixed with polytetrafluoroethylene or foamed tetrafluoroethylene, or an antibody. Alternatively, polytetrafluoroethylene or foamed tetrafluoroethylene may form a first layer on the medical device, followed by fullerenes forming a second layer.</p><p num="0024">The matrix can be non-covalently or feed-bonded to the medical device. Antibodies may be covalently attached to the matrix using heterologous or allogeneic bifunctional cross-linking reagents.</p><p num="0025">Methods for treating atherosclerosis are also provided. The artery can be either a coronary artery or a peripheral artery such as the femoral artery.</p><p num="0026">Overview The present invention provides methods and compositions comprising coating a medical device, such as a stent or synthetic implant, with a matrix used to coat the medical device. In one embodiment, the matrix incorporates a therapeutically effective amount of at least one type of antibody that promotes adhesion of endothelial cells to medical devices. After adhesion, endothelial cells differentiate and proliferate on the surface of the matrix. The presence of endothelial cells on the medical device reduces restenosis and the development of thrombi after vascular implantation of the medical device.</p><p num="0027">As used herein, the term "antibody" refers to a type of monoclonal or polyclonal antibody, where a monoclonal or polyclonal antibody binds to an antigen or a functional equivalent of that antigen. The term antibody is Fab, F (ab')_２Alternatively, it includes any fragment of an antibody such as an Fc fragment. (Antibodies are 6.022 x 10 per mole of antibody.^２３Contains multiple distinct antibodies equal to one molecule. )</p><p num="0028">As used herein, "therapeutically effective amount of antibody" means the amount of antibody that promotes adhesion of endothelial cells to medical devices. The amount of antibody required to carry out the claimed invention varies depending on the nature of the antibody used. For example, the amount of antibody used will depend on the binding constant between the antibody and the antigen with which it reacts. Methods of determining a therapeutically effective amount of antibody for the use of a particular antigen are known to those of skill in the art.</p><p num="0029">As used herein, "medical device" refers to a device that is temporarily or permanently introduced into a mammal for the prevention and treatment of medical symptoms. These devices include any that is subcutaneously, transdermally, or surgically introduced and left in an organ, tissue, or lumen. Medical devices include stents, coated stents such as those coated with polytetrafluoroethylene (PTFE) or foamed polyterolafluoroethylene (ePTFE), synthetic implants, artificial heart valves, artificial hearts and artificial organs for vascular circulation. Fixtures for connection, venous valves, abdominal aortic aneurysm (AAA) implants, inferior aortic filters, permanent drug infusion catheters, embolic coils, embolic materials used for vascular embolization (eg, PVA foam), And vascular sutures can be mentioned.</p><p num="0030">As used herein, "restenosis" refers to the accumulation of layers of smooth muscle and matrix proteins in the intima of the arterial wall. Blood vessels can become occluded due to restenosis. After PTCA or PTA, smooth muscle from the media and adventitia is not normally present in the intima, but proliferates, migrates to the intima, secretes proteins, and intima contains smooth muscle and matrix proteins. Form an accumulation. This accumulation causes stenosis of the lumen of the artery, reducing blood flow from the stenosis to the distal side. As used herein, "restenosis suppression" refers to suppression of smooth muscle migration and proliferation achieved by prevention of protein secretion to prevent restenosis and the resulting complications.</p><p num="0031">Subjects that can be treated using the methods and compositions of the invention can be mammals, more specifically humans, dogs, cats, pigs, rodents or monkeys.</p><p num="0032">The method of the present invention may be carried out in vivo or in vitro.</p><p num="0033">The term "endothelial cells" refers to endothelial cells at any stage of development from origin to maturity. Fully differentiated endothelial cells can be isolated from veins such as arteries or human umbilical veins, while primordial endothelial cells are isolated from peripheral blood or bone marrow. By incubating the endothelial cells with a matrix-coated medical device that incorporates antibodies or other agents that adhere to the endothelial cells, the endothelial cells are bound to the medical device.</p><p num="0034">The method of the invention may be performed in any artery or vein. Atherosclerosis of any artery, including coronary arteries, subgroin arteries, aortic ileal arteries, subclavian arteries, mesenteric arteries and renal arteries, is included within the scope of the invention. Other types of vascular occlusion, such as those resulting from dissecting aneurysms, are also included in the present invention.</p><p num="0035">The medical device may be coated with endothelial cells after insertion into a blood vessel. Alternatively, the medical device may be coated with endothelial cells prior to insertion into the medical device. In either case, the presence of endothelial cells on the luminal surface of the medical device suppresses or prevents restenosis and thrombi.</p><p num="0036">Endothelial cells Human umbilical vein endothelial cells HUVEC are described in Jaffe et al. , J. Clin. Invest. , 52: 2745-2757, 1973 (incorporated herein by reference), obtained from the umbilical cord. Briefly, treatment with collagenase strips cells from the blood vessel wall, 10% low endotoxin fetal bovine serum, 90 μg / ml preservative-free porcine heparin, 20 μg / ml endothelial cell proliferation supplementation (ECGS), Culture in a gelatin-coated tissue culture flask in M199 medium containing glutamine and antibodies.</p><p num="0037">Primordial endothelial cells are referred to by methods such as Asahara et al. (Isolation of putative gential endothelial cells for angiogenesis), Science 275: 964-967, 1997, as used herein. Is isolated from human peripheral blood according to the above. Magnetic beads coated with an antibody against CD34 are incubated with human peripheral blood. After incubation, bound cells can be eluted and cultured in M-199 containing 20% fetal bovine serum and bovine brain extract (Clonetics, San Diego, CA). Cells are characterized by fluorescent antibodies against CD45, CD34, CD31, Flk-1, Tie-2 and E-selectins.</p><p num="0038">Using conventional methods, any animal that contains any cloned gene encoding a protein such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nitrogen oxide synthase (NOS). Transfection of endothelial cells with an expression vector (eg, a mammalian expression vector and transfection kit (transduction) commercially available from Stratagene, San Diego, CA). Kit) (see transfection kit). For example, purified porcine primordial endothelial cells are phase I of angiogenic gene therapy for the treatment of coronary artery disease using a method such as Rosengart (direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA). Evaluation of the 6th month of the clinical trial (Six-month angiogenesis of a phase I trial of angiogenic gene therapy for the treatment of the colony artery disease using direct intracardial administrative of an adenovirus vector expressing the VEGF121 cDNA). Ann Surg. 230 (4): 466-470 (1999), incorporated herein by reference) transfected by vascular endothelial growth factor (VEGF) using an adenovirus expression vector expressing VEGF cDNA. Will be done.</p><p num="0039">antibody Monoclonal antibodies useful in the methods of the invention are the standard techniques of Kohler and Milstein, a continuous culture of fusion cells secreting antibodies of a given specificity (Continuous cultures of fused cells secretting antibody: predefineed skill. It can be produced according to 497, 1975 (incorporated herein by reference). The endothelial cells can be used as an immunogen to produce monoclonal antibodies against the endothelial cell surface antigens.</p><p num="0040">Monoclonal antibodies against endothelial cells are prepared by injecting mice or rats with Hubeck or purified primordial endothelial cells. After sufficient time, the mice are sacrificed to obtain spleen cells. Generally, the spleen cells are immortalized by fusing the myeloma cells or lymphoma cells with the spleen cells in the presence of a nonionic detergent such as polyethylene glycol. The resulting cells contain a fusion hybridoma, which is grown on a selective culture medium such as HAT culture medium and the residual cells are grown on such culture medium using limiting dilution conditions. Cells are grown in a suitable container (eg, a microtiter well) and the supernatant is screened for monoclonal antibodies with the desired specificity, ie, reactivity to endothelial cell antigens.</p><p num="0041">There are various techniques for collecting ascites fluid after increasing the yield of monoclonal antibodies, such as injecting hybridoma cells into the peritoneal cavity of a mammalian host that receives the cells. If an insufficient amount of monoclonal antibody is collected in the ascites fluid, the antibody is collected from the host's blood. There are various conventional methods for the isolation and purification of monoclonal antibodies to isolate them from other proteins and other contaminants.</p><p num="0042">Fab, F (ab') of these monoclonal antibodies_２Alternatively, useful binding fragments of anti-endothelial cell monoclonal antibodies such as Fc fragments are also included within the scope of the invention. Antibody fragments are obtained by conventional techniques. For example, useful binding fragments may be prepared by peptidase digestion of the antibody with papain or pepsin.</p><p num="0043">The antibodies of the invention relate to IgG class antibodies derived from mouse sources, but this does not imply any limitation. Antibodies and antibodies that are functionally equivalent to the antibodies (whether they are mouse sources, mammalian sources including humans, other sources, or combinations thereof), and Other classes (including isotypes of such classes) such as IgM, IgA, IgE, etc. are included within the scope of the invention. In the case of antibodies, the term "functional equivalence" means that each of the two different antibodies binds to the same antigenic site on an antigen, in other words, the antibodies compete to bind to the same antigen. The antigen may be on the same or different molecules.</p><p num="0044">In one embodiment, a monoclonal antibody that reacts with the endothelial cell surface antigen CD34 is used. Anti-CD34 monoclonal antibodies bound to solid supports have been shown to capture primordial endothelial cells from human peripheral blood. After capture, these primordial cells are capable of differentiating into endothelial cells (Asahara et al. 1997. Isolation of putative progenitor endothelial cells for angiogenesis). Science 275: 964-967). Hybridomas that produce monoclonal antibodies to CD34 can be obtained from the American Type Tissue Collection (Rockville, MD). In another embodiment, a monoclonal antibody that is reactive with the endothelial cell surface antigen Flk-1 or Tie-2 is used.</p><p num="0045">Polyclonal antibodies that are reactive against endothelial cells isolated from the same species that accept medical device implants can also be used.</p><p num="0046">Stent As used herein, the term "stent" means any medical device that, when inserted into the lumen of a blood vessel, dilates the cross-sectional lumen of the blood vessel. The term "stent" includes coated stents, such as those coated with PTFE or ePTFE. In one embodiment, it is a stent delivered percutaneously to treat coronary artery occlusion or to occlude an incision or aneurysm in splenic, carotid, iliac and patellar vessels. Including. In another embodiment, the stent is delivered to a venous vessel. The stent can be composed of a polymeric or metallic structural element (a matrix is applied onto the structural element), or the stent can be a composite material of a polymer mixed matrix. For example, deformable metal wire stents such as those disclosed in US Pat. No. 4,886,062 (Wiktor, incorporated herein by reference) can be used. Self-expanding stents of elastic polymeric materials such as those disclosed in International Patent Publication No. 91/12779 (Intraluminal Drug Elution Prosthesis, incorporated herein by reference) may also be used. it can. Stents may also be made from stainless steel, polymers, nickel-titanium, tantalum, gold, platinum-iridium, or Elgiloy and MP35N and other iron materials. The stent can be delivered through the body lumen by the catheter to the treatment site where the stent is released from the catheter and in direct contact with the lumen wall of the blood vessel to expand the stent. It will be apparent to those skilled in the art that other self-expanding stent designs (eg, elastic metal stent designs) can be used with the antibodies and matrices of the invention.</p><p num="0047">Synthetic graft The term "synthetic implant" means any artificial prosthesis with biocompatible properties. In one embodiment, it includes a synthetic implant made from Dacron (polyethylene terephthalate, PET) or Teflon (ePTFE). In another embodiment, the synthetic implant is composed of polyurethane. In a third embodiment, the synthetic implant is composed of an inner layer of mesh polycarbonate urethane and an outer layer of mesh dacron. It will be apparent to those skilled in the art that any biocompatible synthetic implant can be used with the antibodies and matrices of the invention (Bos et al. 1998. Small Diameter Vascular Prosthesis: Small-Diameter). vascular Processes: Current Matrix, Archives Physio Biochem. 106: 100-115, incorporated herein by reference). Synthetic implants are used for vascular incisal anastomosis or for the bypass of an affected vascular segment. Can be used.</p><p num="0048">matrix (A) Synthetic material The matrix used to coat the stent or synthetic implant may be selected from synthetic materials such as polyurethane, segmented polyurethane-urea / heparin, polyL-lactic acid, cellulose ester or polyethylene glycol.</p><p num="0049">(B) Natural material The matrix may be selected from naturally occurring substances such as collagen, laminin, heparin, fibrin, cellulose or carbon. The first requirement of the matrix is that it is sufficiently elastic and flexible to remain unbroken on the exposed surface of the stent or synthetic implant.</p><p num="0050">(C) Fullerene The matrix may also contain fullerenes (the term "fullerene" includes multiple fullerene molecules). Fullerenes are molecules in the carbon cage. The number of carbon molecules in fullerene species varies from about C60 to about C100. Fullerene can be produced by a process known to those of skill in the art, for example, by laser evaporation of carbon, carbon heating in an arc, or burning of hydrocarbons in sooting flames (Patent et. 5,292,813). Al.), Incorporated herein by reference, and produced by high temperature reactions of carbon elements or carbon-containing species according to US Pat. No. 5,558,903 (Bushan et al), incorporated herein by reference). Will be done. In each case, carbonaceous deposits or soot are produced. From this soot, various fullerenes can be obtained by extraction with a suitable solvent such as toluene. Fullerenes are separated by known methods, especially by high performance liquid chromatography (HPLC). Fullerenes may be synthesized or Dynamic Enterprises (Dynamic) Enterprises, Ltd. , Berkshire, England), or may be obtained commercially from Southern Chemical Group, LLC, Tucker, Georgia.</p><p num="0051">Fullerenes are applied to surfaces by a wide variety of methods, including sublimation, laser evaporation, sputtering, ion beams, spray coating, immersion coating, roll-on or brush coating, as disclosed in US Pat. No. 5,558,903. It may be deposited.</p><p num="0052">An important feature of fullerenes is their ability to form "activated carbons". The electronic structure of fullerenes is a system in which π orbitals are superposed so that a large number of bonded electrons coexist around the surface of the molecule (Chemical and Engineering News, Apr. 8, 1991, page 59, by reference). Incorporated herein). In the form of activated carbon, fullerenes are a considerable van der for weak interactions. Waals) Shows power. The adsorptive nature of the fullerene surface may be further modified by itself for the purpose of inducing specific cell membrane interactions. For example, a specific molecule (eg, lectin or antibody) having a chemical property that selectively binds to a specific cell membrane of a specific cell type or a specific component of the cell membrane can be adsorbed on the fullerene surface. The fullerene surface may also be chemically modified to impart a group that is specifically reactive with the cell membrane (eg, an oxidant or reductant). The binding of various molecules to the fullerene surface may be engineered to create a surface that selectively binds various cell types such as endothelial cells, fibroblasts, primary explants or T cell subpopulations. (US Pat. No. 5,310,669 (Richmond et al.), Incorporated herein by reference, Stephen r Wilson, "Biological Objects of Fullerenes", Kadish et al. , "Fullerenes: Its Chemistry, Physics and Techniques" (Fullerenes: Chemistry, Physics and) Technology), eds. , John Wiley &amp; Sons, NY2000, incorporated herein by reference).</p><p num="0053">Fullerenes may also form nanotubes that incorporate other atoms or molecules (Liu et al. Science 280: 1253-1256 (1998), incorporated herein by reference). The synthesis and preparation of carbon nanotubes is known in the art (see US Pat. No. 5,753,088 (Olk et al) and US Pat. No. 5,641,466 (Ebbsen et al.)). Incorporated herein by. Molecules such as proteins may also be incorporated within the carbon nanotubes. For example, on nanotubes, after cutting the ends of the nanotubes, enzymes such as Zn_２Cd_２-Metallothionein, cytochrome C and C3, and β-lactamase may be loaded (Davis et al. Inorganica Chim. Acta 272: 261 (1998); Cook et al. Full Sci. Tech. 5 (4): 695 (4): 1997), both incorporated herein by reference).</p><p num="0054">Three-dimensional fullerene structures can also be used. US Pat. No. 5,338,571 (Mirkin et al., Incorporated herein by reference) (i) chemically modifies fullerenes to provide bond-forming species and (ii) substrates. The surface of the fullerene is chemically treated to provide an effective bond-forming species for covalent bonding with the fullerene bond-forming species in the solution, and the (iii) treated substrate surface is contacted with the modified fullerene solution. The three-dimensional multilayer fullerene structure formed on the substrate surface is disclosed by forming a covalently bonded fullerene layer on the treated substrate surface.</p><p num="0055">(D) Matrix application to medical devices The matrix should adhere firmly to the surface of the stent or synthetic implant. Preferably, this is achieved by applying the matrix to a continuous thin layer. Each layer of the matrix may incorporate the antibody. Alternatively, the antibody may be applied only to the layer in direct contact with the vascular lumen. The various types of matrices may be applied continuously in a continuous layer. The antibody may be covalently or non-covalently coated on the matrix after application of the matrix to the stent.</p><p num="0056">To coat medical devices such as stents, stents are soaked or sprayed with a solution of a matrix of moderate viscosity. After each layer is applied and before the next layer is applied, the stent is dried. In one embodiment, the thin paint matrix coating does not exceed a total thickness of 100 μm.</p><p num="0057">For example, a suitable matrix coating solution is available under sterile conditions in 3 ml (ml) of chlorophosm, such as Poly D-lactic acid (Boehringer Inc., Ingelheim, Germany) R203). Prepared by dissolving 480 milligrams (mg) of drug carrier. However, as a rule, any biodegradable (or non-biodegradable) matrix that is blood and tissue compatible (biocompatible) and capable of dissolving, dispersing or emulsifying is on the medical device after application. May be used as a matrix if it is subject to relatively quick drying to a sticky lacquer or paint coating.</p><p num="0058">For example, coating a stent with fibrin is known to those of skill in the art. Muller et al. ), U.S. Pat. No. 4,548,736 (incorporated herein by reference), fibrin is coagulated by contacting thrombin with fibrinogen. Preferably, in US Pat. No. 3,523,807 (incorporated herein by reference) issued to Gerendas to improve the mechanical properties and biostability of the implanted device. As described or as described in published European Patent Application No. 0366564 (incorporated herein by reference), the fibrin in the fibrin-containing stents of the present invention is present during coagulation. It has factor XIII and calcium. Preferably, the fibrinogen and thrombin used to make fibrin in the present invention are the same as the species to which the stent will be implanted to avoid any interspecific immune response (eg, human anti-cow). It is of animal or human species. The fibrin product can be in the form of a fine fibrin film produced by casting a combination of fibrinogen and thrombin onto a film and then osmotically passing through a semipermeable membrane to remove water from the film. In European Patent Application No. 0366564, the substrate, which is preferably highly porous or has a high affinity for either thrombin or fibrinogen, is contacted with fibrinogen and thrombin solutions. Polymerization of fibrinogen on the surface of medical devices results in the formation of a fibrin layer. The multiple layers of fibrin applied by this method make it possible to provide a fibrin layer of any desired thickness. Alternatively, fibrin can first be solidified, then ground into a powder, mixed with water, and embossed into the desired shape in a hot die (US Pat. No. 3,523,807). Increased stability can also be achieved with molded fibrin by contacting fibrin with a fixative such as glutaraldehyde or formaldehyde. Manufacture fibrin</p><p num="0059">If the synthetic implant is coated with collagen, a method of preparing collagen and forming it on a synthetic implant device is described herein by reference to US Pat. No. 5,851,230. Known as described in). This application describes a method of coating a synthetic implant with collagen. The method of adhering collagen to a porous substrate typically involves applying a collagen dispersion to the substrate, drying it and repeating this process. Collagen dispersions are typically made by blending insoluble collagen (about 1-2% by weight) into the dispersion at acidic pH (pH in the range 2-4). The dispersion is typically injected into the lumen of the implant by syringe and manually manipulated to cover the entire internal surface with collagen slurry. Excess collagen slurry is removed through one of the open ends of the implant. The coating and drying steps are repeated several times to provide sufficient treatment.</p><p num="0060">In yet another embodiment, the stent or synthetic implant is coated with amorphous carbon. In U.S. Pat. No. 5,198,263 (incorporated herein by reference), a method for producing fast, low temperature deposits of amorphous carbon films in the presence of fluorinated gases or other halogenated gases. Is described. Deposition by the method of the present invention can be carried out below 100 ° C., including outside room temperature, using a high frequency plasma assisted chemical deposition process. Amorphous carbon films produced using the methods of the invention are well adhered to many types of substrates, including, for example, glass, metals, semiconductors and plastics.</p><p num="0061">The fullerene-graft, binding of the fullerene moiety to the reactive amino group site of the amine-containing polymer to form the amine-containing polymer may be carried out as described in US Pat. No. 5,292,813. .. Chemical modifications in this way allow the direct incorporation of fullerenes into the stent. In another embodiment, fullerenes may be deposited on the surface of a stent or synthetic implant, as described above (see WO 99/32184 (Leone et al.), By reference herein. To be used in). Fullerenes may also be bound by aldehyde bonds (Yamago et al., "Chemical Derivation of Organic Fullerenes Through Oxidation by Oxidation, Reduction and CO and CC Bond Formation Reactions". , Redox and C-O and C-C Bond Forming Reactions) J Org Chem, 58 4796-4798 (1998), incorporated herein by reference). The C60O may also be attached to the stent directly via the epoxy group on the fullerene. The bond is via a covalent bond to oxygen. This compound and protocol for coupling is commercially available from Bucky USA, Houston, Texas.</p><p num="0062">(E) Addition of antibody to matrix Antibodies that promote the adhesion of primordial endothelial cells can be covalently or non-covalently incorporated into the matrix. The antibody may be incorporated into each layer of the matrix by mixing the matrix coating solution with the antibody. Alternatively, the antibody may be covalently or non-covalently coated on the final layer of the matrix applied to the medical device.</p><p num="0063">In one embodiment, the antibody is added to a solution containing the matrix. For example, a Fab fragment on an anti-D34 monoclonal antibody is incubated with a human fibrinogen-containing solution at a concentration of 500-800 mg / ml. It will be appreciated that the concentration of anti-CD34Fab fragments varies and one of ordinary skill in the art can determine the optimum concentration without undue experimentation. The stent is added to the Fab / fibrin mixture and the addition of concentrated thrombin (at a concentration of at least 1000 U / ml) activates fibrin. A polymerized fibrin mixture containing Fab fragments incorporated directly into the resulting matrix is pressed thinly (less than 0.12 cm) on the surface of the stent or synthetic implant. In fact, any type of antibody or antibody fragment can be incorporated in matrix solution in this manner prior to coating the stent or synthetic implant.</p><p num="0064">In another embodiment, the antibody is covalently attached to the matrix. In one embodiment, the antibody is covalently bound to the matrix using a heterologous or homologous bifunctional linker molecule. As used herein, the term "binding" refers to the covalent attachment of an antibody to a matrix by a linker molecule. The use of linker molecules in the context of the present invention typically involves covalently attaching the linker molecules to the matrix after the matrix has been attached to the stent. After covalent binding of the matrix, the linker molecule provides the matrix with several active functional groups that can be used to covalently bind one or more antibodies. FIG. 1 provides a diagram of binding via crosslinked molecules. Endothelial cells 1.01 bind to antibody 1.03 by cell surface antigen 1.02. The antibody is constrained to the matrix 1.05-1.06 by the cross-linking molecule 1.04. The matrix 1.05 to 1.06 adheres to the stent 1.07. The linker molecule may be attached to the matrix either directly (ie, by a carboxyl group) or by known coupling chemistry (eg, esterification, amidation, and acylation). The linker molecule may be a diamine or triamine functional compound that is attached to the matrix by the direct formation of an amide bond and provides an amine functional group that can be used to react with the antibody. For example, the linker molecule can be a polyamine functional polymer such as polyethyleneimine (PEI), polyallylamine (PALLA) or polyethylene glycol (PEG). Various PEG derivatives, such as mPEG-succinimidyl procrate, or mPEG-N-hydroxysuccinimide, are available from Shearwater Corporation, Birmingham, Alabama, along with a protocol for covalent bonding. Available (also Weiner et al. "Effects of polyethylene glycol spacers on antigen capture by immobilized antibody" (Influence of a poly-ethylene glycol spacer on antibody capsule by immobilized antibodies). J. Biochem. Biophyss. Methods 45: 211-219 (2000), also incorporated herein by reference). It will be appreciated that the choice of particular coupling agent depends on the type of antibody used and such choice can be made without undue experimentation. Mixtures of these polymers can also be used. These molecules contain multiple pendant amine functional groups that can be used to surface-immobilize one or more antibodies.</p><p num="0065">The antibody may be attached to a C60O fullerene layer deposited directly on the surface of the stent. The cross-linking agent may be covalently attached to the fullerene. The antibody is then bound to the cross-linking agent, which in turn binds to the stent. FIG. 2 provides a diagram of C60O coupling. Endothelial cells 2.01 are bound to antibody 2.03 via cell surface antigen 2.02 and subsequently to matrix 2.04 covalently or non-covalently. Matrix 2.04 is covalently attached to stent 2.06 via C60O (2.05).</p>

<figref num="1">The antibody is covalently bound to the matrix by the cross-linking molecule.</figref><figref num="2">The figure of the C60O molecule which fixes a matrix is shown.</figref>

[Experimental example] The present invention is described in the following experimental details section. These sections are set forth below for the sake of understanding the invention, but are not intended or intended to limit the invention in any case, as described in the claims above. Should not be interpreted.

Example 1 Adhesion of human endothelial cells to CD34fab-coated stent Materials and methods 1 cell Hubec (Human Umbilical Cord Endothelial Cells) (HUVEC) is described in Jaffe, EA, "Biology of Endothelial Cells", EA Jaffe, ed., Martinus- Prepared from human umbilical cord by the method of Nijoff, THE Hague (1984), incorporated herein by reference), 20% bovine fetal serum (FCS), L-glutamic acid, antibiotics, 130 μg / ml heparin and 1 It is cultured in culture medium 199 supplemented with 2 mg / ml endothelial cell growth aid (Sigma-Aldrich, St. Louis, MO).

Primordial endothelial cells are isolated from human peripheral blood by methods such as Asahara et al. (Isolation of Putative protease endothelial cells for angiogenesis), Science 275: 964-967. The monoclonal anti-CD34 antibody is bound to magnetic beads and incubated with the leukocyte fraction of whole human blood. After incubation, bound cells are eluted and cultured in M-199 containing 20% fetal bovine serum and bovine brain extract (Clonetics, San Diego, CA). Cells are characterized by CD45, CD34, CD31, Flk-1, Tie-2 and E-selectin.

2 Stent coating A. R stents produced by Orbus International B.V. (Leusden, THE Netherlands), along with a Fab fragment of an anti-CD34 monoclonal antibody, with human fibrinogen (Sigma, St Louis, MO) 500-800 mg / ml. Incubate and polymerize fibrinogen by adding 1000 units / ml of thrombin. After incubation of the stent with a polymerized fibrin mixture containing an anti-CD34 monoclonal antibody fragment, the fibrin is compressed into a thin film (less than 0.012 cm) against the R-stent. A thin fibrin membrane containing a Fab fragment is washed 3 times with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) at room temperature.

B. Alternatively, the R stent is coated with mPEG-succinimidyl propionate Shearwater Corporation (Birmingham, Alabama). The succinimidyl group is reacted with the anti-CD34 monoclonal Fab fragment (Fab-PEG coating R stent) according to the production instructions to form a stable amide bond between the PEG derivative and the Fab fragment.

3 Endothelial cell junction assay Fibrin-anti-CD34 Fab coated R stent, or Fab-PEG coated R stent, 5% CO_２With isolated HUVEC or isolated primordial endothelial cells at a cell concentration of 100,000 to 1,000,000 cells / ml in M199 containing 0.5% BSA at 37 ° C. in a humid atmosphere. Incubate with. Prior to incubation with the stent, HUVEC or primordial endothelial cells, [^３H] -label with thymidine for 24 hours. After incubating the stent and labeled endothelial cells coated with fibrin and Fab anti-CD34 for 4 to 72 hours, the stent is removed from the solution and washed 5 times with M199 containing 0.5% BSA. The bound endothelial cells are removed by trypsin treatment to bind the labeled endothelial cells to the stent.^３H] -Evaluated by thymidine scintillation counting. As a negative control, a stent coated with fibrin alone or an uncoated stent,^３H] -Incubate with thymidine-labeled endothelial cells. Results are statistically evaluated using t-test to establish characteristic binding. Fibrin-coated stents incorporating a monoclonal anti-CD34Fab fragment show a significant increase in endothelial cell binding compared to uncoated stents.

Example 2 Proliferation of human endothelial cells On a CD34Fab coated stent Endothelial cell proliferation assay Fibrin-coated R stents incorporating anti-CD34Fab fragments are incubated with Hubec or primordial endothelial cells in M199 containing 0.5% BSA for 4-72 hours. After incubating the stent with Hubec or primordial endothelial cells, the stent was washed 5 times with M199 containing 0.5% BSA, and then [^３H] -Incubate with thymidine. [[^３H] -Thymidine uptake is assessed on washed and collected Hubec or primordial endothelial cells (cells are collected using trypsin). Hubec or primordial endothelial cell proliferation on fibrin-coated stents is compared to endothelial cell proliferation on a standard microtiter dish. Proliferation of Hubec or primordial endothelial cells on fibrin-coated stents equals or exceeds that of endothelial cells in microtiter dishes.

Example 3 Production of monoclonal antibodies reactive with Hubec and primordial endothelial cells BALB / c mice were subjected to 1.5 × 10 in PBS.^６Hubeck, or 1.5x10^６Primordial endothelial cells are immunized intraperitoneally at intervals of 3-4 times every 2-4 weeks, 1.5 x 10 3 days before splenocytes are removed.^６Hubeck, or 1.5x10^６Attack with primordial endothelial cells. Spleen cell suspensions are prepared and fused with myeloma NS1 / 1AG4.1 to grow hybridomas and clone them. In order to improve the efficiency of hybridoma growth and cloning, 10% endothelial cell-conditioned culture (HUVEC) is included in the culture medium. First, hybridoma culture supernatants are tested for reactivity with Hubec or primordial endothelial cells by immunofluorescence flow cytometry (FACS). Briefly, Hubeck (1.5x10)^４) Or primordial endothelial cells (1.5 × 10)^４) Is incubated with undiluted hybridoma supernatant (30 minutes, 4 ° C.), washed and washed with fluorescein-isothiocyanate (FITC) -sheep F (ab')._２Incubate with anti-mouse Ig (100 μg / ml). After the final wash, endothelial cells are examined for monoclonal antibody binding by immunofluorescent flow cytometric analysis. Positive hybridoma supernatants are screened for human melanoma cell line MM-170 to eliminate non-endothelium-specific mAbs. Screening of monoclonal antibodies in human tumor cell lines and panels of human leukocytes, monocytes, neutrophils, erythrocytes and platelets further confirms endothelial specificity.

Example 4 Study of porcine balloon injury Antibody-coated stents are transplanted in young York shear pigs weighing 25-30 kg. Animal management follows the Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, reviewed 1985). After an overnight fast, the animals are sedated with ketamine hydrochloride (20 mg / kg). After induction of anesthesia with thiopental (12 mg / kg), the animal is intubated and connected to a vent to which a mixture of oxygen and nitrous oxide (1: 2 [vol / vol]) is administered. Sustain anesthesia with 0.5-25% by volume isoflurane. Intramuscular injection of 1,000 mg of a mixture of penicillin G procaine and penicillin G benzathine (streptomycin) provides antibiotic prophylaxis.

Under sterile conditions, an arteriotomy of the left coronary artery is performed and a 9F-introduction needle sheath is placed in the left coronary artery. All animals are given intravenous heparin sodium 7,500 IU and 100 mg acetylsalicylic acid. An additional 2,500 IU bolus of heparin is regularly administered throughout the procedure to maintain active coagulation time of 300 seconds or longer. An 8F lead catheter is introduced through the carotid artery sheath and passed to the origin of the iliac artery. After administration of 1 mg of isosorbide dinitrate, angiography is performed and the images are analyzed using a quantitative coronary angiography system. A 3F-embolic resection catheter is inserted into the common femoral artery and passed distally from the selection zone for stent implantation. The embolic incision balloon is inflated 0.5 mm larger than the arterial segment and pulled twice to expose the blood vessel. Immediately after exposure, a fibrin-coated stent incorporating a Fab fragment of the monoclonal antibody is inserted through an induction catheter and placed in the exposed area of the femoral artery. Animals are sacrificed both 3 days and 8 weeks after stent implantation. As mentioned above, the animal is first sedated and anesthetized. The stented femoral segment is externally transplanted and then placed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 ° C. for 48 hours. A rectangular section of the vessel wall is removed for further processing for electron microscopic evaluation of the surface coverage of endothelial cells. This portion of the stented vessel is placed in 0.15 cacodelate buffer and further immobilized with 2.5% glutaraldehyde in 0.15 M cacodylate. Next, 1% OsO_４And 50 mM ferricyanide (K_３[Fe (CN)_６There is a reduction in thrombosis with a Heparin Coating Palma-Schazz stent in normal porcine coronary arteries, where the tissue is post-fixed and further processed with 0.1 M cacodile buffer containing. (Reduction in). thrombotic events with heparin-coated Palmaz-Schatz stents in normal coronary arteries), Organization 93 423-430, incorporated herein by reference).

Residual sections of the stented arterial section are as described by van Beausecom et al. (Cardiovasc Pathol 5: 69-76 (1996), incorporated herein by reference). Soak with methyl methacrylate in three states. An electrically rotating microtome (HM-350, Microm) using a stainless steel disposable knife to occupy an embedded artery segment with a stent in place. Cut into 3-5 μm thick sections with GmbH, Munich, Germany). On a chrome-aluminum-coated slide, the sections are stretched on a hot plate at 40 ° C. with 60% 2-butoxyethanol and 10% ethanol in water. The sections are covered with a plastic film to remove excess butoxyethanol-ethanol mixture and placed in an oven at 40 ° C. overnight to dry the slides. Subsequently, the plastic is removed from the section for 30-60 minutes in an equal volume of xylene-chloroform solution (deplasticize). The prepared sections are then subjected to standard staining procedures for light microscopy. Statistical data is expressed as the mean ± standard error (SD) of the mean for separate experiments. Statistical significance is determined by analysis of variance (ANOVA) and Fisher's PLSD test (Stat View 4.01, Brain Power, Inc, Calabasas, Calif). Paired t-test (Stat View) for femoral artery treated and untreated plot data 4.01) is used. A p-value less than 0.05 is considered to be a statistically significant difference between the mean values. Animals treated with stents incorporating anti-porcine endothelial cell monoclonal Fab fragments show increased endothelial cell coverage and a significant reduction in restenosis compared to controls with transplanted uncoated stents.

Example 5 Transfection of porcine primordial endothelial cells Porcine primordial endothelial cells are isolated from porcine peripheral blood by a method such as Asahara (Isolation of Putative protease endothelial cells for angiogenesis), Science 275: 964-967. .. The monoclonal anti-CD34 antibody is bound to magnetic beads and incubated with the leukocyte fraction of whole porcine blood. After incubation, bound cells are eluted and cultured in M-199 containing 20% fetal bovine serum and bovine brain extract (Clonetics, San Diego, CA). Cells are characterized by CD45, CD34, CD31, Flk-1, Tie-2 and E-selectin.

For example, purified porcine primordial endothelial cells are used in Phase I clinical trials of angiogenic gene therapy for the treatment of coronary artery disease using a method such as Rosengart (direct intracardiac administration of an adenovirus vector expressing VEGF121 cDNA). 6-month evaluation of the (six-month assessment of a phase I trial of angiogenic gene therapy for the treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing tHE VEGF121 cDNA) .Ann Surg.230 (4): 466 -470 (1999), incorporated herein by reference) are transfected with vascular endothelial growth factor (VEGF) using an adenovirus expression vector expressing VEGF cDNA.

Transfected, VEGF-expressing purified porcine primordial cells are subjected to a porcine femoral artery model after balloon injury and stent implantation using a dual balloon chamber infusion catheter (Cordis Corp) as described in Example 4. Inject and isolate the stented portion of the femoral artery. Restenosis in VEGF-transfected porcine primordial cell-injected balloon angioplasty stent-treated porcine is compared to porcine infused with untransfected porcine primordial endothelial cells. Expression of VEGF in reinjected primordial porcine endothelial cells results in the occurrence and reduced severity of restenosis in anti-CD34 coated stents.

Example 6 Preparation of antibody bound with aminosilane PEO Stent preparation The stent is made of 316L stainless steel and is cleaned and passivated by first washing in an anionic cleaner with an ultrasonic cleaner, then immersed in hot nitric acid with stirring and finally finally. Rinse with deionized water.

The derivatized stent is prepared as follows: The stent is immersed in a 2% mixture of N- (2-aminoethyl-3-aminopropyl) trimethoxysilane in 95% ethanol for 3 minutes and removed. After air-drying at room temperature, it is cured at 110 ° C. for 10 minutes.

Coupling derivatized stents of polyethylene glycol (PEG) spacers were placed in 100 ml of 0.1 M MES buffer containing 10 mM dicarboxymethyl-PEG, 500 mg of EDC was added and 25 ° C. with constant stirring for 2 hours. Incubate at.

Restraint antibody Mouse anti-CD34IgG_１By immersing the stent in 150 ml of 0.1 M MES buffer (pH 4.5) in which 1.0 mg of antibody is dissolved, the antibody against endothelial cells is PEG-functionalized by a one-step carbodiimide coupling reaction. Immobilize on a stent and incubate at 25 ° C. for 2 hours. The stent is removed from the solution and rinsed 5 times with 50 ml (pH 7.2) of phosphate buffered saline having 0.02% Tween 20.

Reagents include N- (2-aminoethyl-3-aminopropyl) trimethoxysilane (DegussA-Huls), MES buffer-morpholine ethanesulfonic acid buffer (Sigma, st.Louis, MO), EDC-1- Ethyl-3- (3-dimethylaminopropyl) carbodiimide (Sigma, st.Louis, MO), dicarboxymethyl-PEG-dicarboxymethyl-poly (ethylene glycol) [MW 3400] (Shearwater, Huntsville, AL). Be done.

Although several different embodiments of the present invention have been described, the present invention is not intended to be limited to these embodiments and is defined by those skilled in the art in the spirit of the invention. And it is intended that changes and modifications can be made without departing from the scope.