CN102363051B - Medical device with coating for capturing genetically-altered cells and methods of using same - Google Patents

Abstract

The present invention relates to a medical device with a coating for capturing genetically-altered cells and a method of using the same, and particularly provides therapeutic and drug delivery systems. The coating on the medical device may be a biocompatible matrix comprising at least one type of ligand, such as antibodies, antibody fragments, other peptides and small molecules, which recognize and bind the target cells. The therapeutic and/or drug delivery systems may be provided with a signal source such as activator molecules for stimulating the modified cells to express and secrete the desired marker and therapeutic gene products.

Description

There is medical treatment device and the using method thereof of the coating of cell that can be capturing genetically-altered

Patent application of the present invention is that international application no is PCT/US2005/015555, international filing date is on April 29th, 2005, the application number that enters the China national stage is " 200580012581.X ", and denomination of invention is the divisional application of the application for a patent for invention of " having medical treatment device and the using method thereof of the coating of cell that can be capturing genetically-altered ".

The application requires to be filed in the U.S. Provisional Patent Application No.60/566 on April 30th, 2004, and 829 and be filed in the U.S. Provisional Patent Application No.10/835 on April 30th, 2004,767 priority.

Technical field

The present invention relates to the medical treatment device for patients with implantation blood vessel or hollow organ, for example be used for the treatment of the support, stent graft through applying of various diseases, synthetic blood vessel graft, cardiac valve, conduit and vascular repair screen (vascular prosthetic filter).Particularly, the present invention relates to the cated medical treatment device of tool on the surface contacting with blood, described coating can be used for cell capture in the surface of described device through engineered.The cell of catching can form monolayer and can be used for many therapeutic use on the surface of described device, for example, in drug delivery system and/or treatment angiopathy.For example, the cell that is incorporated into the medical treatment device of implantation can be from the natural endothelial progenitor cells of circulating and/or genetically modified cell in vitro, and described cell has molecule or the material of part or whole body therapeutic effect in patient's expression in vivo or secretion.

Background of invention

Atherosclerosis in disease and cancer are two kinds of main causes that cause in the world dead and disability.Atherosclerosis produces relevant with the lip-deep fatty spew of lumen of artery.These fatty spews cause lumen of artery narrow in cross-section.Finally cause the Oligemia at diseased region far-end, cause being subject to the tissue generation ischemia injury of this tremulous pulse blood supply.

Coronary artery is to heart blood supply.In the U.S., coronary atherosclerosis or coronary heart disease (CAD) are modal life-threatening severe chronic diseases, suffer from this sick number and exceed 1,100 ten thousand people.The society that coronary atherosclerosis causes and economic loss are considerably beyond most of other diseases.Coronary arterial lumen is narrow in myocardium impact, first causes angina pectoris, is then myocardial infarction, finally causes death, in these patients, have 300,000 people above before arriving at hospital with regard to dead.(" harrisonShi internal medicine principle", Harrison ' s Principles of Internal Medicine, the 14th edition, 1998).

For coronary atherosclerosis, can adopt through percutaneous transluminal coronary Transluminal Angioplasty (PTCA) and treat.The annual above PTCA of 400,000 examples that implements of the U.S. performs the operation.In PTCA, balloon catheter is inserted in peripheral arterial, be advanced to the coronary artery being blocked along Arterial system.Then make airbag inflation, this section of arterial strutted, flatten the fatty speckle that blocks this place, thereby increase the blood flow in this impaired tremulous pulse cross section of flowing through.But this treatment can not make impaired coronary artery maintain enduringly open configuration conventionally.Nearly 50% the needs of patients of accepting PTCA treatment was accepted repetitive therapy to correct restenosis coronarius in 6 months.The narrow restenosis that is medically called that this PTCA of acceptance artery occurs once again.Acute restenosis relates to resilience and the shrinkage of blood vessel.After blood vessel resilience and shrinkage, there is propagation to tackle by the caused arterial injury of PTCA in middle level smooth muscle cell.The various inflammatory factor that the propagation part of smooth muscle cell is discharged by damage location mediates, and this class inflammatory factor comprises: thromboxane A2, platelet derived growth factor (PDGF) and fibroblast growth factor (FGF).Adopted multiple different technologies to overcome restenosis problem, these technology comprise: with various medicaments, patient is treated or with rack mechanical keep tremulous pulse to open.(" HarrisonShi internal medicine principle ", Harrison ' s Principles of Internal Medicine, the 14th edition, 1998).

Prove that support is to overcome effective method in the various Therapeutic Method of restenosis.Support is to be placed in the metal supporting frames that produces normal blood vessels inner chamber in diseased arteries section.Can prevent the resilience of this tremulous pulse and the blood vessel graft then occurring by propping up to be placed in impaired tremulous pulse section.Support also can prevent that this tremulous pulse from peeling off along medial part.Be greater than by make arterial lumens keep with support the inner chamber that independent employing PTCA forms, reduced nearly 30% restenosis.But although obtain such success, support still can not be eliminated restenosis completely.(Suryapranata etc., 1998." for through select patients of acute myocardial infarction in Coronary stenting and the stochastic comparison of balloon angioplasty ", Randomized comparison of coronary stenting with balloon angioplasty in selectedpatients with acute myocardial infarction circulation, 97:2502-2502).

Stricture of artery also can occur in other blood vessels outside coronary artery, comprises tremulous pulse under aorta bone tremulous pulse, groin, the dark femoral artery in distally, Yuan Ce popliteal tremulous pulse, tibial artery, subclavian artery and Mesenteric artery etc.Atherosclerosis (PAD) prevalence of peripheral arterial depends on the impaired concrete region of anatomy, and standard used is blocked in diagnosis.Traditionally, doctor adopts intermittent claudication method of testing to determine whether and has PAD.But this method may have been underestimated the actual sickness rate of this disease in crowd greatly.The sickness rate of PAD is different with the age, and the sickness rate of older individuals PAD increases.According to U.S.'s state hospital leave hospital investigation data estimation, have every year 55,000 routine male and 44,000 routine women to be diagnosed as first chronic PAD; And have 60,000 routine male and 50,000 routine women to be diagnosed as first acute PAD.91% acute PAD case all relates to lower limb position.In PAD patient, the ratio of the CAD that occurs together may exceed 50%.In addition,, in PAD patient, cerebrovascular disease sickness rate increases.

Can adopt percutaneous transluminal balloon angioplasty (PTA) to treat PAD.Adopt PTA and in conjunction with support, can reduce restenosis sickness rate.But, adopting the postoperative effect that obtains such as the medical treatment device of support, the effect obtaining not as the revascularization (, adopting vein or reparation conduit) of employing standard.(" surgery principle", Principles of Surgery, the volumes such as Schwartz, the 20th chapter, " arterial disease ", arterial disease, the 7th edition, McGraw-Hill Health Professions Division, New York 1999).

Preferably adopt the method treatment PAD that sets up bypass, walk around obstructing arterial section with graft in the method and set up bypass.(" principles of surgery", Principles of Surgery, the volumes such as Schwartz, the 20th chapter, " arterial disease ", Arterial Disease, the 7th edition, McGraw-Hill Health Professions Division, New York 1999).Described graft can comprise: autogenous vein section (for example, saphena) or synthetic graft (graft of for example, preparing with polyester, politef (PTFE) or expanded polytetrafluoroethyltoe (ePTFE) or other various polymeric materials).Its postoperative vessel open rate depends on multiple different factor, and these factors comprise: the intracavity footpath of bypass graft, as the synthetic material type of graft, and flow out position.But even if adopted bypass graft, neointimal hyperplasia and thrombosis remain serious problem.For example, under the groin of employing ePTFE bypass graft, arterial bypass is after 3 years, and the vessel open rate of burst-popliteal bypasses is 54%, and the bypass of thigh-shin is only 12%.

Therefore, obviously need to improve support performance, synthetic bypass graft and other long-term blood contact surfaces and/or device performance, further to reduce sickness rate and the case fatality rate of CAD and PAD.For example, can adopt and can make the method for blood vessel radial dilatation (outside or positivity is moulded again) compensate the carrying out property growth of atheromatous plaque, thereby postpone the development of blood flow limited narrow (flow-limiting stenosis).

For support, the method having adopted is to carry out drug delivery medical device by various anticoagulants or anti-restenosis agent, to reduce thrombosis and restenosis.For example, seem to suppress restenosis by the migration and the propagation that are suppressed to myofibroblast by radioactive substance impregnated stents.(U.S. Patent number 5,059,166,5,199,939 and 5,302,168).But irradiate the blood vessel for the treatment of and can cause the edge restenosis problem that patient is serious.In addition, irradiation is inhomogeneous to being encroached on management of blood vessels.

Or, also adopt chemical agent drug delivery medical device, for example heparin, phosphocholine, rapamycin and taxol, all these medicines all show can extenuate thrombosis and/or restenosis.Can obviously reduce in short-term the thrombosis of animal model although heparin and phosphocholine show, this class medicament seems not have long-time effect to prevention of restenosis.In addition, heparin may bring out thrombocytopenia, causes serious thrombus complication, for example apoplexy.Therefore, in the practice mode of this treatment restenosis, with enough the heparin for the treatment of effective dose or the support of phosphocholine loading are also infeasible.

Adopt synthetic graft to treat in many ways to reduce postoperative restenosis and thrombosis.(Boe etc., 1998." small diameter vascular shifting plant prothesis: present situation ", Small-Diameter Vascular Graft Prostheses:Current Status, archives Physio.Biochem,106:100-115).For example, it is reported, it is suitable with ePTFE graft that polyurethane synthetic (for example, porous Merlon urethanes) reduces the effect of restenosis.Also adopt the modifying surface of radio frequency glow discharge to described graft, so that poly-, dibenzoate graft has been fluoridized.Also adopt biomolecule to flood synthetic graft.But none can significantly reduce thrombosis or restenosis incidence rate over a long time these methods.

Endotheliocyte (EC) layer is a kind of important component of normal blood vessels wall, and it provides the interface between blood flow and blood vessel wall surrounding tissue.Endotheliocyte also can participate in physiological activity, comprising: angiogenesis, inflammatory process and prevent thrombosis (Rodgers GM.FASSEB J 1988; 2:116-123).In recent years research discovery, endotheliocyte is except composition vascular system, and postnatal EC and endothelial progenitor cells (EPC) also circulate in peripheral blood.(Asahara T etc.Science, 1997; 275:964-967; Yin AH, etc.Blood, 1997; 90:5002-5012; Shi Q, etc.Blood, 1998; 92:362-367; Gehling UM, etc., Blood, 2000; 95:3106-3112; Lin Y etc., J Clin Invest, 2000; 105:71-77).It is believed that EPC can migrate to the impaired endodermis of blood circulation region, comprises wound and ischemic injury position (Takahashi T etc., Nat Med, 1999; 5:434-438).In normal adult, the EPC concentration in peripheral blood is 3-10/mm ³(Tkahashi T etc., Nat Med, 1999; 5:434-438; Kalka C, etc., Ann Thorac Surg, 2000; 70:629-834).Nowadays verified, every one-phase that blood vessel is replied for damage is all subject to the impact (if not controlling) of endothelial tissue.It is believed that; impaired and settle the rapid Reconstruction of The Function endodermis of blood vessel section of support can be by the ability of various materials of the harmful effect of the barrier to the circulating cells factor, anti-tampon and generation that they have protection lower floor layer of smooth muscle cells is provided, and contribute to the severe complication that prevents that these are potential.(Van Belle etc., 1997; " the interior dermatoplasty of support ", Stent Endothelialization, circulation, 95:438-448; Bos etc., 1998." small diameter vascular shifting plant prothesis: present situation ", Small-Diameter Vascular Graft Prostheses:Current Status, archives Physio. biochem.106:100-115).

After implant frame, by local delivery vascular endothelial cell growth factor (VEGF, a kind of endotheliocyte mitogen), can promote growth (Van Belle etc., 1997 of endotheliocyte at rack surface; " the interior dermatoplasty of support ", Stent Endothelialization, circulation,95:438-448).Bring out required action effect although can apply the saline solution of recombinant protein growth factor VEGF at damaged part, VEGF can send with passage balloon catheter after implant frame.But technique is unsatisfactory, this is can not produce consistent effect because of the effect that it is confirmed that single delivery dosage is too low.Thereby this method can not accurately be reappeared effect at every turn.

Also adopted endothelial cell seeding synthetic material graft, but the clinical effectiveness of plantation endotheliocyte is conventionally all very poor, be that postoperative tube chamber opens the low (Lio etc. of rate, 1998, " new ideas of blood capillary transplantation and new material: endothelial cell seeding and the gene therapy of reparation property graft ", New concepts and Materials in Microvascular Grafting:Prosthetic Graft Endothelial Cell Seeding and Gene Therapy, Microsurgery, 18:263-256), its reason is that cell is failed to be properly attached on graft and/or because isolated operation causes cell and lost its EC function most probably.

Thereby in the adjusting of sticking, growing and breaking up of vascular injury site endotheliocyte, original position endothelial cell growth factor (ECGF) and environmental condition are vital.Accordingly, for restenosis and other angiopathy, be necessary to develop the new method and composition for applying the medical treatment device including support and synthetic graft, on implanting device, form functional endothelium with promotion and acceleration, thereby on the inner chamber of target vessel section or implantation, form continuous EC monolayer, suppress thus new intima hyperplasia.

About diseases such as cancers, current most of medicine can produce whole body systemic reaction to patient, owing to using the medicine of conventional oral or intravenous form, not only can affect cancerous cell, also can affect any fissility cell (dividing cell) in body.In many cases, such as, due to the character of disease and the character of medicine (dissolubility, body internal stability, bioavailability etc.) of need treatment, general administration is ineffective.In the time of general administration, medicine is defeated and be distributed into the body area including normal structure through blood circulating.At diseased region, originally drug level low and invalid, can bring up to toxic level, and in non-lesion region, the existence of medicine causes bad side effect after frequent drug administration.In some cases, medicine is easy to be subject to metabolic degradation after administration.Therefore, often obtain drug effect time expand by improving drug dose, this just causes the increase of normal structure system burden and the increase with patient's correlative charges.In other cases, due to the toxic and side effects of some active drug, its treatment potential can not be brought into play completely.

Therefore, many effort have been done and have improved effect and the targeting of drug delivery system.For example, the advantage that adopts liposome delivery medicine to have be conventionally their can prolong drug circulation time in blood, reduce side effect, reduce drug degradation, extend therapeutical effect after each administration, reduce the demand to administration frequency and reduce the amount of required medicine by the concentration of free drug in restriction blood flow.But the effect that current available liposome system demonstration delivers drugs into target site is in vivo limited.Referring to Kaye etc., 1979, Poznansky etc., 1984, United States Patent (USP) 5,043,165 and United States Patent (USP) 4,920,016.

For realizing the high-efficiency delivery to therapeutic compound, developed the viral vector that can mix transgenic DNA, but the successful quantity of clinical practice is still limited.The successful number obtaining in external and animal model, propose gene transfer technique to combine with cell therapy.Ex vivo gene transfer is entered in various cell types likely to prove to shift and have larger treatment feasibility than direct body carrier.Referring to Kohn etc., 1987, Bilbao etc., 1997, and Giannoukakis etc., 2003.

Recently, develop localized drug delivery carrier, for example bracket for eluting medicament (drug eluting stent, DES).Referring to United States Patent (USP) 6,273,913, United States Patent (USP) 6,258,121 and United States Patent (USP) 6,231,600.But bracket for eluting medicament of the prior art is subject to being permitted multifactorial restriction, for example, the time of drug type, the medication amount that discharge and release medicine.The other factors that need to consider about bracket for eluting medicament is that medicine and other support apply for example, interaction between composition (polymeric material) and the character (for example, integrity after lipotropy, molecular weight, sterilizing and activity and effect and toxicity) of individual drugs.As for the polymeric material in bracket for eluting medicament, what must consider is type, polymerization ratio, the ability of drug loading and the biocompatibility of this polymer of polymer, and the compatibility of drug-polymer (for example, the pharmacokinetics of medicine).

In addition, the drug dose in bracket for eluting medicament is preloaded, can not realize the drug dose of individual condition and demand is regulated.As for pharmaceutical release time, once bracket for eluting medicament starts to discharge medicine after implanting immediately, and the real-time release that can not realize ideal.

Therefore, in the urgent need to developing a kind of effectively general and local drug delivery system, to overcome the defect of current available technologies.The present invention be just to provide a kind of peace and and controlled way carry out the system of part or systemic delivery medicine.

Summary of the invention

An object of the present invention is to provide a kind of method of curative drug delivery system and treatment patient disease.This treatment or drug delivery system comprise the cated medical treatment device of tool, this coating is by containing that at least one class can be identified and forming in conjunction with the substrate of the part of target cell, and described target cell is for for example: the mammalian cell of endothelial progenitor cells or hereditary change and be subjected to the mammalian cell of the hereditary change of single or dual transfection.

Medical treatment device of the present invention can be any implantable patient's device.For example, in one embodiment, described device is the device that can insert intravascular space or hollow organ, as support, stent graft, cardiac valve, conduit, vascular repair screen, artificial heart, external and built-in left ventricular assist device (LVAD) and synthetic vessel graft, they are used for the treatment of following disease: for example cancer; Angiopathy, comprises restenosis, atherosclerosis, thrombosis, angiemphraxis; Or these install other any purposes covering.

In one embodiment, coating on described medical treatment device comprises: biocompatible matrix and at least one substrate or part, described substrate or ligand specificity identification for example, in conjunction with target cell (endothelial progenitor cells), for example to prevent or to treat restenosis, or the mammalian cell that makes hereditary change adheres to the surface of described device, for example to treat vascular remodeling and cancer.

In addition, the coating of described medical treatment device optionally comprises at least one engineered gene expression that can regulate hereditary change cell and the reactive compound of secretion.The example of the activator of energy activated compounds includes but not limited to: chemical molecular and peptide, for example somatomedin.In the embodiment that comprises at least one compound in coating, stimulus object, anakmetomeres or compound can be used for irritation cell expression and/or secrete at least one treating the therapeutant of disease.

In one embodiment, coating on medical treatment device comprises the biocompatible matrix with outer surface, described outer surface is treated at least one part (for example, the combination of antibody, antibody fragment or antibody and antibody fragment) of effective dose or at least one can be in conjunction with the molecule of the engineered labelling of genetically altered cell surface for adhering to.The identification of antibody of the present invention or antibody fragment and in conjunction with the antigen on cell membrane or target cell surface or specificity through genetically engineered cell surface marker, thereby make cell be fixed on the surface of described device.In one embodiment, described coating optionally comprises at least one compound of fixing for stimulating endothelial CFU-GM of effective dose, if target cell is circulation CFU-GM, can promote the formation of maturation, functionalization endothelium, if or target is the hereditary change cell in surfaces of medical devices, can stimulates the cellular expression of combination and secrete required gene outcome.

Medical treatment device of the present invention can be for implanting the organ that comprises tract or any device of body part, also can be but be not limited to: support, stent graft, synthetic blood vessel graft, cardiac valve, conduit, vascular repair screen, pacemaker, pacemaker lead, defibrillator, in hole, ovum garden patent (PFO) every closing device, vascular clamp, vascular aneurysms dead lock, hemodialysis graft, hemodialysis catheter, chamber diverter, aortic blood tuberculation graft device or assembly, venous valve, suture, blood vessel anastomosis clamp, remain-type vein or ductus arteriosus, vagina vasorum and drug conveying mouth.According to the difference of device, described device can be made from a variety of materials.For example, support of the present invention can be made with rustless steel, Nitinol (NiTi) or evanohm and Biodegradable material.Synthetic blood vessel graft can be made by crosslinked PVA hydrogel, politef (PTFE), expanded polytetrafluoroethyltoe (ePTFE), porous type high density polyethylene (HDPE) (HDPE), polyurethane and poly terephthalic acid vinyl acetate or Biodegradable material.

The biocompatible matrix that forms described medical treatment device coating is including but not limited to synthetic material, for example, and polyurethane, block polyurethane-carbamide/heparin, Poly-L-lactide, cellulose esters, Polyethylene Glycol, polyvinyl acetate, glucosan or gelatin; And/or naturally occurring material, the for example basement membrane composition of collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin and so on, heparin, fibrin, cellulose and amorphous carbon or fullerene (fullerene).

In an embodiment of the invention, in described medical treatment device, contain the biocompatible matrix that comprises fullerene.In this embodiment, the carbon number of fullerene can be about C ₂₀-C ₁₅₀, more specifically, described fullerene is C ₆₀or C ₇₀.Fullerene of the present invention also can be arranged in nanotube (nanotube) on the surface of medical treatment device.

In an embodiment of the invention, described part is put on to the surface that medical treatment device contacts with blood, described part energy specific recognition in conjunction with the lip-deep epi-position of required component or target cell in circulating.In one embodiment, described part be specifically designed as on the cell membrane by only identifying hereditary change cell through genetically engineered labelled molecule, and can only identify and in conjunction with the mammalian cell of hereditary change.The combination of target cell is fixed on this cell on the surface of device.

In one embodiment, select the part for the cell in conjunction with hereditary change in surfaces of medical devices according to the cell membrane labelled molecule through genetically engineered.That is to say, described part is only combined with cell membrane labelled molecule or the antigen of being expressed by the extrachromosomal genetic element that offers this cell, thereby makes the part in described surfaces of medical devices only identify genetically modified cell.In this way, only have genetically modified cell just to can be incorporated into the surface of medical treatment device.For example, if described mammalian cell is endotheliocyte, described part can be at least one antibody, antibody fragment or their combination; Produce specifically described antibody for the lip-deep specific target epi-position of target cell or labelled molecule.In this one side of the present invention, described antibody can be monoclonal antibody, polyclonal antibody, chimeric antibody or humanized antibody, it is by only identification the endotheliocyte in conjunction with this hereditary change with the surface markers intermolecular reaction of the endotheliocyte of hereditary change, and regulates thus this cell so that it adheres to surfaces of medical devices.Antibody of the present invention or antibody fragment covalently or non-covalently can be connected in to the surface of substrate, or pass through the outermost layer of the covalently bound substrate to coated medical devices of linkers.In this embodiment, for example, monoclonal antibody also can comprise Fab or F (ab ') ₂fragment.The fragment that antibody fragment of the present invention comprises any size, for example, has retained antibody recognition macromole and micromolecule in conjunction with target antigen characteristic.

In another embodiment, the mammiferous antigen that antibody of the present invention or antibody fragment capable are identified specifically and combination is treated, and their specificity does not depend on cell line.In one embodiment, for example, in treatment restenosis, contain specific cell membrane marker when cell is not genetically modified and divide the period of the day from 11 p.m. to 1 a.m, described antibody or fragment can be selected and specifically in conjunction with the surface antigen of circulation endothelium progenitor cell, for example CD133, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Muc-18 (CD146), CD130, stem cell antigen (Sca-1), stem cell factor 1 (SCF/c-Kit part), Tie-2, MHC (for example, H-2K ^kand HAD-DR).

In another embodiment, the coating of described medical treatment device comprises at least above-mentioned biocompatible matrix of one deck, and this substrate is adhered at least one outer surface natural or synthesized micromolecule for the treatment of effective dose containing being useful on.Described micromolecule can for example be identified, the endothelial progenitor cells in treatment of restenosis, and with its interaction, thereby and by this cell be fixed on apparatus surface form endodermis.Described micromolecule can be used for and this medical treatment device therapeutic alliance various diseases, and can be derived from various sources (for example cell component, as fatty acid, protein, nucleic acid, saccharide etc.), and can produce result or the effect identical with antibody with the lip-deep AI of endothelial progenitor cells.Of the present invention this on the one hand, the coating on medical treatment device also can inclusion compound, for example, be connected in the above-mentioned somatomedin of this paper in the coating that comprises antibody or antibody fragment.

In one embodiment, the compound (for example, for treatment of restenosis) in coating of the present invention, comprises and can stimulate or promote growth of progenitor cells and differentiation to become any compound of ripe functional endotheliocyte.In another embodiment, described compound is for stimulating genetically modified cellular expression and secreting required gene outcome.For example, compound for the present invention can be somatomedin, somatomedin, transforminggrowthfactor-β1, acid fibroblast growth factor, osteonectin, Ang-1 (Ang-1), ANG2 (Ang-2), insulin like growth factor, granulocyte macrophage colony stimulating factor, PDGF-A A, PDGF-BB, PDGF-A B and the endothelium PAS albumen 1 of for example VEGF (VEGF), basic fibroblast growth factor, platelet induction.

In another embodiment, for example, in the time using the mammalian cell of hereditary change, can be used for irritation cell expresses and secretes activator or the compound through genetically engineered gene outcome, include but not limited to: estrogen, tetracycline and other antibiotic, tamoxifen etc., can offer patient through various route of administration, for example, pass through percutaneous drug delivery with paster and subcutaneous form.

The present invention is also provided for treating the method for various diseases, and described disease is for for example: angiopathy, cancer, vascular remodeling, severe coronary artery disease, atherosclerosis, restenosis, thrombosis, aneurysm and angiemphraxis.In one embodiment, provide a kind of for keep or seal insert blood vessel wall medical treatment device (for example, support or synthetic blood vessel graft, cardiac valve, abdominal aortic aneurysm device and their parts) and set up environment in blood vessel stable state, thus prevent as the method for the excessive neointimal hyperplasia in restenosis.In the atherosclerotic the inventive method for the treatment of, described tremulous pulse can be coronary artery or peripheral arterial (for example femoral artery).Also can treat vein with these technology and medical treatment device.

For the treatment of restenosis, it is a kind of for inducing the engineered method of healing reaction that the present invention also provides.In one embodiment, a kind of method that forms the endodermis merging for rapid induction on the luminal surface at the implanted device of grafting vessel target region is provided, and wherein said endotheliocyte can be expressed nitricoxide synthase and other antiinflammatory and inflammation regulatory factor.The present invention also provides a kind of medical treatment device, it has higher biocompatibility compared with prior-art devices, and can be by reducing or suppressing the differentiation of smooth muscle cell migration, smooth muscle cell and the collagen deposition along the surface of internal cavity of medical treatment device implant site, reduce or suppress excessive neointimal hyperplasia and the restenosis organized.

In one embodiment, method for coated medical devices comprises the following steps: at least one deck biocompatible matrix puts on surfaces of medical devices, and wherein said biocompatible matrix comprises at least one component that is selected from lower group: polyurethane, block polyurethane-urea/heparin, Poly-L-lactide, cellulose esters, Polyethylene Glycol, polyvinyl acetate, glucosan, gelatin, collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose and carbon and fullerene; And to described biocompatible matrix simultaneously or in succession apply: at least one antibody, antibody fragment or their combination for the treatment of effective dose, and the compound of at least one stimulating endothelial cell growth and differentiation.

The present invention also provides a kind of method for the treatment of mammal blood vessel disease, described method comprises: medical treatment device is implanted in mammiferous blood vessel or pipe inner chamber, wherein said medical treatment device is coated with (a) biocompatible matrix, (b) at least one antibody, antibody fragment or their combination for the treatment of effective dose, and (c) at least one compound; Wherein said antibody or antibody fragment capable identification in conjunction with the lip-deep antigen of endothelial progenitor cells, thus endothelial progenitor cells is fixed in stromal surface, and described compound is for stimulating fixing endothelial progenitor cells to form endothelium in surfaces of medical devices.

Treatment/the drug delivery system for the treatment of patient disease is also provided in one embodiment.The mammalian cell that described treatment or drug delivery system contain hereditary change and for the medical treatment device of patients with implantation, the mammalian cell of wherein said hereditary change comprises coding through genetically engineered cell surface marker and the exogenous nucleic acid of at least one therapeutic gene product.In one embodiment, with genetic engineering modified cell described in the suitable transfection carrier in-vitro transfection that comprises exogenous genetic material, for described cell provides required gene.In this embodiment, described cell can be any mammalian cell, no matter be autologous, allochthonous or xenogenesis, and such as endotheliocyte, fibroblast, sarcoplast etc.In this embodiment, described medical treatment device applies with biocompatible matrix, described substrate comprises part, and described part is the antigen on genetically engineered cell membrane labelled molecule or cell surface by combination, and is only incorporated into the mammalian cell of hereditary change.

In treatment of the present invention and/or drug delivery system, the cell of described hereditary change has imported the required gene of at least one Codocyte surface markers molecule or antigen and the gene of at least one coding therapeutic gene product with exogenous genetic material.This system option ground comprises signaling system, for example, can stimulate the mammalian cell expression of hereditary change and/or secrete reactive compound or the molecule of required gene outcome and/or marker gene.

Therefore, in one embodiment, by import described exogenous genetic material in mammal through engineered can specific binding device for codified on the cell membrane labelling of part.For example, when described device is used for inserting intravascular space, this exogenous genetic material should be encoded except offering patient's the cell membrane labelling all not having in any cyclicity cell in auterblood through genetically engineered cell.

A kind of medical treatment device and method through applying that is used for the treatment of various diseases is also provided, and described disease is: for example angiopathy, includes but not limited to atherosclerosis, cancer and rheumatoid arthritis.Medical treatment device of the present invention comprises for the coating that the mammalian cell of hereditary change was caught and fixed to specificity in vivo, and the mammalian cell of described hereditary change is to import when by the described medical treatment device patients with implantation through applying simultaneously or subsequently.

The fixing hereditary change cell of expressing and/or secreting at least one material that is used for the treatment of specific disease or therapeutic agent for fixing is also provided.In this one side of the present invention, for example, in treatment cancer, make described cell (for example, endotheliocyte) that hereditary change occur by import exogenous genetic material in described cell.In one embodiment, in described hereditary material transfered cell core, and it is DNA (for example exchromosomal DNA).Described exchromosomal DNA can be carrier, such as adenovirus vector, plasmid (as gymnoplasm grain), linearity or short dna etc.In one embodiment, described DNA comprises regulation and control/expression cassette of controlling the required labelling of expression and/or therapeutic gene.In one embodiment, described regulation and control box can comprise the controlling element for constructive expression's therapeutic gene, and maybe can comprise can be by the element of needs of patients control or expression.

In one embodiment, the described medical treatment device for patients with implantation comprises coating; Described coating comprises by least one of substrate load can be identified and in conjunction with the part of target cell.In the embodiment of the cell that is hereditary change at cell, described part is only identified and in conjunction with through engineered and add specific cell membrane marker molecule or the antigen of cell.Therefore in this embodiment, part is only identified the mammalian cell of the hereditary change that has imported patient, the mammalian cell of this hereditary change is incorporated on described medical treatment device, expresses and secrete labelled molecule or antigen and at least one therapeutic gene product.

In another embodiment, described treatment or drug delivery system also can comprise activating molecules (activating molecula), and this activating molecules is for stimulating the mammalian cell expression of described hereditary change and/or secreting required therapeutic gene product.In this one side of the present invention, can the compound such as chemical irritant or peptide be supplied with to patient by several different methods, these methods comprise: oral route, warm paster (thermal patch), intravenous, intradermal injection etc.In this embodiment, the mammalian cell of described hereditary change can be autologous or external source, such as mature endothelial cell, fibroblast, myocyte, epithelial cell etc., and the exogenous nucleic acid that they comprise can be exchromosomal DNA.In one embodiment, described DNA provides with carrier format, such as adenovirus vector, naked plasmid dna, linear DNA etc.In one embodiment, described exchromosomal DNA comprises regulation and control box, and the gene of Codocyte membrane antigen and at least one coding are used for the treatment of the gene of the peptide of disease.In aspect of this embodiment, described cell membrane specific gene coding, for example BMP or prostatic cell memebrane protein.

In one embodiment, the gene that described extrachromosomal genetic element comprises the treatment/pharmaceutical product of encoding, these products are for example VEGF and the angiogenesis factor for vascular remodeling, or are used for the treatment of the anti-angiogenesis of cancer.

In another embodiment, provide a kind of method that is used for the treatment of disease.Described method comprises:

The mammalian cell of hereditary change is offered to patient; Described cell comprises coding through genetically engineered cell membrane labelled molecule and at least one therapeutic gene product exogenous nucleic acid;

By the medical treatment device patients with implantation that comprises coating; The substrate of at least one part that described coating has comprised load, wherein this part can identify and in conjunction with on the mammalian cell of described hereditary change through genetically engineered cell membrane labelled molecule, the mammalian cell of wherein said hereditary change is incorporated into medical treatment device, and therapeutic gene product described in expression and secretion.In an embodiment of the invention, described therapeutic genes and gene outcome comprise for example VEGF, angiogenesis factor, anti-angiogenesis and fibroblast growth factor.

The present invention also provides the method for the treatment of patient disease, and described method comprises: the mammalian cell of hereditary change is offered to patient; By medical treatment device patients with implantation; Described medical treatment device comprises coating, described coating comprises load the substrate of at least one part, described part energy specific recognition in conjunction with at least one labelled molecule, for example, receptor on the mammalian cell of this hereditary change, the mammalian cell of wherein said hereditary change is incorporated on described medical treatment device, comprises the exogenous nucleic acid for expression and secretion therapeutic gene product.

In another embodiment, provide a kind of in vivo cell being raised to the surperficial method of contact blood.Described method comprises provides the surface that is arranged in object blood flow and contacts blood, and the surface of described contact blood is through setting up the surface that the cyclicity target cell in object blood flow can be raised to described contact blood; And the surface of described target cell being raised to described contact blood.In this embodiment, the surface of described contact blood comprises the surface of internal cavity of the medical treatment device of implanting described object.In this embodiment of the present invention, the target cell of for example being raised, on the surface (support or graft) that contacts blood can recover the surface of described device self endothelialization (self-endothelialize) normal endothelial tissue at damaged blood vessels position.The surface of described contact blood can be biodegradable framework maybe can be coated with biodegradable biocompatible materials.In this one side of the present invention, described biodegradable framework will be degraded in position in the time of implantable intravascular, and the newborn endothelium forming on described device inner chamber has been rebuild by the coherent blood vessel of injury, thereby form functional new vessels.

In another embodiment, the present invention includes a kind of dummy, described dummy comprises: (a) have outer surface and the carrier film that contacts blood surface; (b) be coated on the lip-deep ground floor cross-linked polymer compound of contact blood of described carrier film; (c) be coated on the second layer on ground floor, the described second layer contains at least one part in vivo target cell with affinity.

In another embodiment, provide a kind of for generating in vivo the method for self endothelialization graft, described method comprises: it is the framework as blood vessel graft that the function of setting up (a) is provided, described framework has surface of internal cavity and outer surface, and described surface of internal cavity comprises specificity for the part in conjunction with endothelial progenitor cells; (b) described framework is implanted in the blood vessel of object; (c) cyclicity endothelial progenitor cells is raised on the described surface of internal cavity of described framework to form newborn endothelium.

In another embodiment, provide a kind of method for original position generation self endothelialization graft, described method comprises: the surperficial reparation construction with contact blood circulation (a) is provided; (b) described reparation construction is implanted to object; (c) from blood, raise cyclicity cell (for example, the mammalian cell of endothelial progenitor cells and hereditary change) and make on its surface that is attached to described reparation construction, thereby form newborn endothelium thereon.

In another embodiment, the method of original position generation self endothelialization graft is provided, described method comprises: it is the biodegradable framework as interim blood vessel graft that the function of setting up (a) is provided, and described framework has surface of internal cavity and outer surface; (b) by described biodegradable framework implantable intravascular; (c) for example raise, for example, on the surface of internal cavity that circulating cells (mammalian cell of endothelial progenitor cells and hereditary change) makes it to be attached to described dummy (graft, support or biodegradable framework), thereby form newborn endothelium; (d) make outer surface that vascular tissue wraps up described framework to form outside hemostasis blood vessel structure; (e), in vivo under condition, can make described newborn endothelial tissue and outside blood vessel structure form in the time period of functional new vessels, described biodegradable framework is degraded.

In one embodiment, provide a kind of biodegradable framework that forms endothelialization blood vessel graft for original position, described framework comprises: the porous biological degradable carrier film (a) with surface of internal cavity and outer surface; (b) described surface of internal cavity comprises the ground floor being made up of at least one polymerizable compound being coated in described carrier film, wherein said compound can form covalent bond through cross-linking agent self-crosslinking, described covalent bond can be subject to enzymatic lysis or non-enzymatic hydrolysis under condition in vivo, and (c) mammalian cell of Binding in vivo hereditary change is had the part of specificity affinity.

In another embodiment, provide a kind of method for original position generation self endothelialization graft, described method comprises: the surperficial reparation construction with contact patient circulating (a) is provided; (b) described reparation construction is implanted to object or patient; (c) give described patient by the mammalian cell of hereditary change; (d) from blood, raise cyclicity cell (mammalian cell of for example hereditary change) and make on its surface that is attached to described reparation construction, thereby on the surface of described reparation construction, form the cell of one deck hereditary change.

In another embodiment, a kind of method that promotes vascular remodeling is provided, for example increase aortic girth by outside or positivity reconstruct and occupy partly or entirely to compensate because forming that atheromatous plaque causes or the injured rear inner chamber causing because of inner membrance paraplasm of tremulous pulse, thus prevent or suppress damaged blood vessels inwardly or negativity reconstruct.In this embodiment, for example providing as above is covered and can be in conjunction with the support of genetic engineering modified cell by substrate and part, for example, for catching genetically modified autogenous cell (endothelial progenitor cells), these cells can be secreted at least one potent anticoagulant and vasodilation, for example: prostacyclin, as PGE1 2, PG12; Calcitonin-gene-related peptide, as α-CGRP etc.Can be through engineered other product producing by cell, comprise nitric oxide (nitric oxide synthase gene), matrix metalloproteinase, acetylcholine, adenosine, 5-hydroxy tryptamine, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P), adrenomedullin etc.Can adopt its product as vasodilation and/or anticoagulant or there is vasodilation and/or the gene of anticoagulant characteristic, for example, can cause the vasodilation of vascular smooth muscle relaxation.Can the gene (for example prostacyclin synthase gene) of coding vasodilation be offered to endothelial progenitor cells or endotheliocyte by gene transfer technique, described gene transfer technique can be and for example adopts the viral gene of cistron (cistronic) gene constructs to shift, with regard to prostacyclin, for example cistron cyclooxygenase-1/ prostacyclin synthase gene constructs can provide continuous local prostaglandin to send.In this embodiment, prostaglandin local delivery system for example can be used for treatment, cerebral infarction and coronary vessels diseases.Also can be by the positivity reconstruct of blood vessel for example, with making adjustments arteriogenesis (being ripe blood vessel, the formation of be grown up small artery and tremulous pulse) to form the Therapeutic Method of attached blood vessel.

In another embodiment, the suitable cell of bicistronic mRNA carrier transfection of available code vasodilation compound and unique cell surface marker (MHC-I of for example truncate), as fibroblast, endotheliocyte or endothelial progenitor cells, described cell surface marker can for example, by part (being fixed on the antibody on dummy in blood vessel) identification.For example, in the coronary artery of the support patients with implantation that part (as antibody) can be applied, then the cell of hereditary change (endotheliocyte of for example hereditary change) is implanted and need to be treated in the patient of angiopathy.In this embodiment and other embodiment of cell that uses hereditary change, can adopt the technique for gene engineering of standard to utilize for example plasmid vector before implanting cell, exogenous gene to be sent into cell, described plasmid vector can be for example bicistronic mRNA pMACSK ^k.II plasmid vector (Miltenyi Biotec, Germany), it comprises multiple clone site and interested gene can be inserted to the wherein selected marker as mammal cell line used, described interested gene is for example prostacyclin synthase and marker gene (as the MHC I type molecule of truncate, H-2K).

In another embodiment, the exogenous gene delivery system for transfection mammalian cell in being used for the treatment of can comprise, for example, comprise the MHC I type antigen of truncate and the slow virus carrier of vasodilation transgenic (being for example used for the treatment of prostacyclin synthase and/or α-CGRP gene of angiopathy).In this embodiment, describedly want transfected mammalian cell can be autologous endotheliocyte or endothelial progenitor cells, and ligands specific (for example anti-H-2K of the MHC I type antigen of available this truncate of described reparation device ^kantibody) apply.

Accompanying drawing summary

Figure 1A is that antibody passes through corsslinking molecular covalent coupling in the schematic diagram of substrate.Figure 1B is C ₆₀o molecule is anchored on the schematic diagram in substrate.Fig. 1 C is the schematic diagram that the present invention is coated with the support of substrate.

Fig. 2 A is the phase contrast microscope photo that sticks to the endothelial progenitor cells on the slide being applied by fibronectin, contains the cell separating from enrichment medium on described slide.Fig. 2 B is the phase contrast microscope photo of endothelial progenitor cells being attached on the slide that fibronectin applies, the cell separating containing the coated magnetic bead of useful anti-CD34 antibody on described slide.Fig. 2 D and 2F are the microphotograpies of having hatched 7 days and having carried out the endothelial progenitor cells of nuclear staining with PI.From these figure, as respectively to the antibody fluorescence display of Tie-2 (Fig. 2 E and 2G) and VEGFR-2 (Fig. 2 C) antibody response, shown in cellular expression the labelling of mature endothelial cell.

Fig. 3 A and 3B are 2% agarose gel sxemiquantitative RT-PCR photos of the ethidium bromide staining of endothelial nitric oxide synthase (eNOS) and glyceraldehyde phosphate dehydrogenase (GAPDH).Be incubated at after upper 3 day of slide (Fig. 3 B) and 7 days (Fig. 3 A) of fibronectin coating, endothelial progenitor cells starts to express eNOS mRNA.

Fig. 4 A-4E sticks to and cultivate and use iodate the third ingot dyeing together with HUVEC cell: CMDx and anti-CD34 antibody (4A); Gelatin and anti-CD34 antibody (4B); Blank rustless steel disk (4C); The photo of HUVEC on (4E) rustless steel disk that (4D) that CMDx applies and gelatin apply.

Fig. 5 A-5C is the microphotograph of the contrast that applies of the CMDx without antibody of hatching by human blood leukocyte's component.Described cell is with the Anti-KDR antibody dyeing of iodate the third ingot and FITC labelling.Fig. 5 D-5F be hatch by human blood leukocyte's component by the microphotograph that is incorporated into the contrast rustless steel disk that its surperficial gelatin applies without antibody.Described cell is with the Anti-KDR antibody dyeing of iodate the third ingot and FITC labelling.

Fig. 6 A-6C is the microphotograph that is combined with the rustless steel disk of the CMDx substrate coating of anti-CD34 antibody on the surface of hatching with HUVEC.Described cell is with the Anti-KDR antibody dyeing of iodate the third ingot and FITC labelling.Fig. 6 D-6F is the microphotograph that the gelatin coating surface of hatching with HUVECS is combined with antibody rustless steel disk.Described cell is with the Anti-KDR antibody dyeing of iodate the third ingot and FITC labelling.

Fig. 7 is the microphotograph that the surface combination of hatching altogether 24 hours with CFU-GM has the rustless steel disk that the CMDx of antibody applies.Described cell is with the Anti-KDR antibody dyeing of iodate the third ingot and FITC labelling.

Fig. 8 A and 8B are the microphotograpies that the surface combination of hatching altogether 7 days with CFU-GM has the rustless steel disk that the CMDx substrate of anti-CD34 antibody applies.Described cell is with the Anti-KDR antibody dyeing of iodate the third ingot and FITC labelling.

Fig. 9 A and 9B are the microphotograpies that the surface combination of hatching altogether 7 days with CFU-GM has the rustless steel disk that the CMDx substrate of anti-CD34 antibody applies.Described cell is with the anti-Tie-2 antibody staining of iodate the third ingot and FITC labelling.

Figure 10 A-10C is the phase contrast microscope photo of hatching the rustless steel disk of the CMDx coating of 3 weeks in endothelial cell growth culture medium with CFU-GM, has shown ripe endotheliocyte.

Figure 11 is the schematic diagram that is combined with the rack surface of the functional fullerene coating of use of the present invention of CFU-GM.

Figure 12 A-12D is the photo of the sample of the fullerene coating of the anti-CD34 antibody of with or without.Described sample and human leukocyte component are hatched altogether, and with anti-VEGFR-2 antibody staining of iodate the third ingot and FITC labelling.

Figure 13 A-13D is low power and the high magnification micrographs of coronary artery extract histology transverse section, the sample (Figure 13 B and 13D) 4 weeks that described extract has been implanted exposed stainless steel stent (Figure 13 A and 13C) and applied with fullerene.Section hematoxylin-eosin staining.

Figure 14 A-14G is the scanning electron microscope microphotograph of implanting 1 and 48 hours after-poppet extracts of male yorker.The extract of (14B) support that (Figure 14 A) and glucosan/anti-CD34 antibody that glucosan applies applies is latter 1 hour of implantation.Figure 14 C and 14D are depicted as control sample extract, and Figure 14 E-G is the support that glucosan/anti-CD34 antibody applies of implanting latter 48 hours.Figure 14 H-14M is cross section histology's microphotograph of taking from the implantation coronary artery extract of 4 weeks of male yorker: uncoated (blank rustless steel) (14H and 14I), glucosan apply (14L and the 14M) that contrast (14J and 14K) and glucosan/anti-CD34 antibody applies.

Figure 15 A, 15B and 15C are respectively the long support applying without the glucosan-blood plasma of antibody with surface of 18mm of implanting 48 hours, the fluorescence micrograph of the apply/support that anti-CD34 antibody applies of glucosan-blood plasma.

Figure 16 A and 16B are the microphotograpies of the sample of iodate the third ingot and antiagglutinin/FITC coupling.

Detailed Description Of The Invention

The invention provides a kind of implantable medical device (for example support or graft) of coating and for applying the method and composition of this medical treatment device, and by the method for the medical treatment device treatment angiopathy of described coating.The present invention also provides one to be used for the treatment of the method for disease (for example restenosis and cancer), described method comprises the patient who cated tool medical treatment device is implanted to needs treatment, and for described patient provides through genetically engineered mammalian cell, described cell can be incorporated in vivo the surface of described medical treatment device and can produce engineered and required therapeutic agent (for example gene outcome).Figure 1A-1C is the schematic diagram of medical device surface coatings of the present invention.Coating on medical treatment device comprises: biocompatible matrix, it can promote to form the fused cell layer (mammalian cell of for example hereditary change on apparatus surface, as endotheliocyte or fibroblast) for example, can prevent restenosis and/or thrombotic anti-angiogenesis or anticoagulant or produce the product that can suppress neointimal hyperplasia to regulate or to produce required therapeutic effect in patient, to produce.In one embodiment, the substrate that coating on described prosthetic device comprises contains synthetic or naturally occurring material, in described material, comprise and can promote cyclicity cell (mammalian cell of for example hereditary change, as endotheliocyte, CFU-GM or stem cell) to adhere at least one antibody for the treatment of effective dose and at least one compound (for example somatomedin) of the growth of energy stimulating endothelial cell and differentiation of medical treatment device.Implanting after described device, the cell transformation that is attached to described apparatus surface becomes functional cell layer ripe and that merge, for example, form endothelium at described medical treatment device surface of internal cavity.The endotheliocyte fused layer existing on medical treatment device, for example, can reduce implant site restenosis and thrombosis.

As used herein, " medical treatment device " refers to temporarily or forever introduces in mammal to prevent or to treat the device of a certain medical science condition of illness.These devices comprise for example, any device in the inner chamber that is placed in organ, tissue or organ (tremulous pulse, vein, ventricle or atrium) of introducing by subcutaneous, percutaneous or operation.Described medical treatment device can comprise: support, stent graft, the support applying (is for example coated with politef (PTFE), expanded polytetrafluoroethyl,ne (ePTFE) or other natural or synthetic coating), or synthetic blood vessel graft, artificial heart valve, artificial heart and can connect the organ of reparation and the fixture of systemic vascular, vein valve, abdominal aortic aneurysm (AAA) graft, postcava screen sheet, permanent infusion administering catheter, spiral shell volume embolus (embolic coil), the embolism materials using in the time that blood vessel embolism forms (for example, crosslinked PVA hydrogel), blood vessel suture, vascular anastomosis fixture, myocardial vascular forms support and/or other various conduits more thoroughly.

The coating of the medical treatment device forming with the compositions and methods of the invention can stimulate the mammalian cell layer that produces fusion on described apparatus surface in vivo.For example, in the time that provided ligand binding endotheliocyte makes to form functional endodermis on its blood contact surface at described device, in this surfaces of medical devices, form endothelial layer, thereby prevent restenosis, and can regulate the local chronic inflammatory reaction and the thrombosis complication that are caused by the implantation of described medical treatment device.

The substrate that covers medical treatment device can be made up of synthetic material, and for example, polymerism gel foam, as the hydrogel of being made up of polyvinyl alcohol (PVA), polyurethane, Poly-L-lactide, cellulose esters or Polyethylene Glycol.In one embodiment, in the synthetic material of preparation substrate, can comprise very hydrophilic compound, for example dextran compound.In another embodiment, described substrate is made up of naturally occurring material, for example collagen, fibrin, elastin laminin, tropoelastin and/or amorphous carbon.Described substrate also can comprise multilamellar, and for example ground floor can be made up of synthetic material or naturally occurring material, and the second layer can contain for example part (as antibody).Can in succession arrange in turn for each layer, ground floor directly contacts with medical treatment device (support or synthetic material graft) surface, and the second layer has a surface directly to contact with ground floor, and another surface contacts with intravascular space.

Described substrate also can comprise at least one somatomedin, cytokine, vasodilation, anticoagulant etc.Can stimulating endothelial cell the somatomedin of propagation and differentiation for for example: VEGF (VEGF) and isoform thereof, basic fibroblast growth factor (bFGF), the somatomedin (PIGF) of platelet induction, transforminggrowthfactor-β1 (TGF. β 1), acid fibroblast growth factor (FGF), osteonectin, Ang-1, ANG2, insulin like growth factor (ILGF), PDGF-A A (PDGF-AA), PDGF-BB (PDGF-BB), PDGF-A B (PDGF-AB), granulocyte macrophage colony stimulating factor (GM-CSF) etc., or its functional fragment, they can be used in the present invention.Vasodilation comprises: prostacyclin, α-CGRP etc.

In another embodiment, described substrate can comprise fullerene, and the carbon number of described fullerene is about C ₂₀-C ₁₅₀.Described fullerene also can be arranged in nanotube, wherein can mix molecule or protein.Described fullerene substrate also can be applicable to rustless steel, PTFE or ePTFE surfaces of medical devices, then this layer is carried out to functionalization and at its surface-coated antibody and somatomedin.Or, can be at first coated with PTFE or ePTFE layer on rustless steel medical treatment device for example, and then apply second layer fullerene, add antibody and somatomedin.

This substrate can mode non-covalent or covalency be attached on medical treatment device.Various antibody and somatomedin can by different base or with the difunctional cross-linking reagent of base, covalent attachment be in this substrate.Can adopt standard technique that somatomedin is added in this substrate together with antibody or after antibodies.

As used herein, term " antibody " refers to a class monoclonal, polyclone, humanization or mosaic antibody or their combination, wherein, described monoclonal, polyclone, humanization or chimeric antibody and a kind of antigen maybe the functional equivalents of this kind of antigen combine.Term antibody fragment comprises that any antibody fragment that has same effect and effect with antibody is (as Fab, F (ab ') ₂deng), and can be any size, i.e. macromole or micromolecule.(include most antibody of antibody molecule separately, be equivalent to 6.022 × 10 ²³individual molecule/mole antibody).

For example, in one embodiment, available biocompatible matrix drug delivery medical device or synthetic graft, this substrate comprises and can regulate circulating cells (mammal therapeutic cells and the endothelial progenitor cells of for example hereditary change) to adhere to antibody, antibody fragment or their combination of described surfaces of medical devices.For example, the cell membrane labelled molecule of antibody recognition of the present invention binding specificity, the endothelial progenitor cells surface antigen and/or the membrane molecule that are for example produced by the mammalian cell of hereditary change in circulating, form functional cell layer (for example functional endothelial tissue) thereby described cell is fixed on the surface of device.In one embodiment, described antibody comprise can with the monoclonal antibody of following substance reaction (identification in conjunction with): the mammalian cell surface molecule of hereditary change, endothelial progenitor cells surface antigen, or CFU-GM or stem cell surface antigen, for example vascular endothelial growth factor receptor-1,-2 and-3 (VEGFR-1, the isoform of VEGFR-2 and VEGFR-3 and VEGFR receptor family), Tie-1, Tie2, CD34, Thy-1, Thy-2, Muc-18 (CD146), CD30, stem cell antigen-1 (Sca-1), stem cell factor (SCF or c-Kit part), CD133 antigen, VE-cadherin, P1H12, TEK, CD31, Ang-1, Ang-2 or the antigen of expressing on described cell surface.In one embodiment, can use and a kind of antibody of antigen reactive single type.Or, the multiple different antibody for different endothelial progenitor cells surface antigens can be mixed and adds in substrate.In another embodiment, adopt the mixture of monoclonal antibody, improved endothelium formation rate by targeting specific cell surface antigen.In this embodiment, for example anti-CD34 of use capable of being combined and anti-CD133, maybe can share these and any or several antigen group listed above for example, in the stromal surface being attached on medical treatment device (support or graft).Also available antibodies, antibody fragment and/or their combination apply described medical treatment device.

As used herein, the antibody of effective dose " treatment " refers to and can promote cell attachment in the antibody consumption of medical treatment device, and wherein said cell is for example: the mammalian cell of natural or hereditary change, comprises endotheliocyte, CFU-GM or stem cell.Implement antibody amount required for the present invention according to the character of antibody used and difference.For example, the amount of antibody used depends on binding constant and/or the affinity between antibody and the antigen of reaction with it.Those of ordinary skill in the art knows the treatment effective dose of how to confirm for the antibody of concrete antigen.

As used herein, term " compound " refers to any material that can stimulate the mammalian cell expression of hereditary change and/or secretion therapeutic gene product.

As used herein, term " somatomedin " refers to can irritation cell (for example hereditary change or unaltered endotheliocyte, stem cell or CFU-GM) grows and is divided into peptide, protein, glycoprotein, lipoprotein or their fragment or variant or the synthetic molecule of ripe functional endotheliocyte.Thereby ripe endotheliocyte can be expressed nitricoxide synthase nitric oxide is released into tissue.Following table 1 has been listed some somatomedin that can be used for applying described medical treatment device.

Table 1

As used herein, " VEGF " refers to any isoform of listed VEGF in table 1, unless can it be differentiated by this sequence number with work or letter abbreviations specifically to term.

As used herein, term " somatomedin for the treatment of effective dose " refers to and stimulates or inducing specific cell mass (for example natural or modified endotheliocyte, CFU-GM or stem cell) growth and differentiation, thus the maturation that formation is merged and the somatomedin consumption of functional cell layer (for example endotheliocyte forms functional endothelial tissue at medical treatment device surface of internal cavity).Implement somatomedin consumption required for the present invention different with the binding kinetics between character and somatomedin and its receptor in target cell of somatomedin used.For example, 100 μ g VEGF have shown and can adhere to medical treatment device and form endothelium fused layer by stimulating endothelial cell.Those of ordinary skill in the art knows for example, treatment effective dose for the somatomedin of irritation cell (, endotheliocyte) growth and differentiation of how to confirm.

As used herein, " neointimal hyperplasia " refers to the bad increase of smooth muscle cell proliferation in blood vessel wall and/or apposition.As used herein, " restenosis " refers to that the recurrent of intravascular space is narrow.Blood vessel can block because of restenosis.After PTCA or PTA, from media and adventitia and the smooth muscle cell not conventionally being present in inner membrance breed, move to inner membrance secretory protein, in inner membrance, form gathering of smooth muscle cell and stromatin.This gathers and has caused the Oligemia that arterial lumens is narrow, flow to stenosis far-end.As used herein, " inhibition restenosis " refers to the inhibition of smooth muscle cell migration and propagation and prevents protein secreting simultaneously, thereby prevent restenosis and the complication causing thereof.

The object of available medical treatment device of the present invention, method and composition treatment is mammal, comprising: people, Canis familiaris L., cat, pig, horse, Rodents and monkey.

It is interior or external that Therapeutic Method of the present invention can be applicable to body.

Term " endothelial progenitor cells " refers to derived from bone marrow, blood or local organization the endotheliocyte in any stage of development (the functional endotheliocyte from CFU-GM or stem cell to maturation), and they are non-malignant cells.

The medical treatment device that can be described coating provides genetically modified mammalian cell, for example can for example, from vitro tremulous pulse or vein (human umbilical vein), separate the endotheliocyte of the differentiation of the hereditary change obtaining, they have carried out hereditary change with required nucleic acid construct thing in vitro, and endothelial progenitor cells is separable from peripheral blood or bone marrow.In one embodiment, can be by endotheliocyte and the medical treatment device that has applied substrate be hatched jointly, and described endotheliocyte can be combined with described medical treatment device, wherein said substrate has been mixed antibody and optional at least one somatomedin or other and can be attached to the part of endotheliocyte.In another embodiment, described endotheliocyte can be the endotheliocyte of conversion.The endotheliocyte of transfection can comprise the carrier of expressing somatomedin or other peptide or protein, and these somatomedin, peptide or protein can directly or indirectly suppress thrombosis, restenosis or other any therapeutic outcome.

In another embodiment, the mammalian cell of available mammalian expression vector transfection endotheliocyte or other type-stable (for example fibroblast), described carrier comprises coding and is suitable for the protein of special-purpose or any clone gene of peptide.For example, can build described carrier makes it contain expression cassette, the gene that described expression cassette comprises coding platelet derived growth factor (PDGF), fibroblast growth factor (FGF) or nitricoxide synthase (NOS), and available conventional method builds described expression cassette, can be provided by the commercially available source of goods.(referring to for example, purchased from Stratagene, San Diego, the mammalian expression vector of CA and transfection reagent box).For example, according to the method for Rosengart etc., use the adenovirus expression carrier of VEGF expression cDNA, with the pig endothelial progenitor cells of VEGF (VEGF) gene transfection purification (" be used for the treatment of and give the assessments in six months that I phase of the angiogenesis gene therapy of the adenovirus vector of VEGF expression 121 cDNA tests in the direct cardiac muscle of coronary heart disease ", 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, include in herein as a reference).In this embodiment, the source of described mammalian cell can be autologous, allochthonous or xenogenesis.Once transfection foreign DNA or the rna expression box and described cytogenetics is changed that contains required gene, the tissue culture technique of available standards is cultivated described cell.The frozen expression secrete the cell sample of required gene in liquid nitrogen of available standards technology.Before use, the tissue culture technique of available standards regrows freezing cell.In the time implanting described device, the mammalian cell of hereditary change given in implant site part or give patient through intravenous or intra-arterial, preferably after the medical treatment device of implanting described coating, giving.Transformant also can comprise and gives before this cell of patient for accurately detecting and differentiate labelling or the reporter gene of described cell.

Can on any tremulous pulse or vein, implement the present invention and treat the method for angiopathy.In the scope of the invention, comprise the atherosclerosis of any tremulous pulse, described tremulous pulse comprises tremulous pulse under coronary artery, groin, aorta iliac artery, subclavian artery, Mesenteric artery and renal artery.The present invention also comprises the angiemphraxis of other type, and what for example dissecting aneurysm caused blocks.

The mammiferous method that treatment suffers from angiopathy comprises: the medical treatment device of coating is implanted to described patient's organ or blood vessel, for example, in revascularization, implanted the support applying.Once enter in body, endothelial progenitor cells is by identification and for example, be trapped on the surface of support of described coating in conjunction with cellular antigens (mammalian cell of genetic modification), or is trapped in by the combination of independent antibody or antibody and other part the CFU-GM surface being present in described device coating.Once CFU-GM is attached to substrate, the somatomedin in described coating just promotes endothelial progenitor cells growth and the differentiation of new combination and forms at the surface of internal cavity of described support the functional endothelial tissue of maturation merging.Or, before the described medical treatment device of implantation, can for example, apply described medical treatment device with the mammalian cell (endotheliocyte) of natural or genetic modification in vitro, these cells can be the CFU-GM, stem cell or the mature endothelial cell that separate from blood samples of patients, bone marrow or blood vessel.In both cases, it is required or through engineered function that the functional cell existing on described medical treatment device surface of internal cavity can produce, for example, suppress or prevent neointimal hyperplasia and thrombosis.

Endotheliocyte

In some embodiments, according to (J.Clin.Invest., 52:2745-2757,1973 such as Jaffe, this include in herein as a reference and for test) method, can obtain human umbilical vein's endotheliocyte (HUVEC) from people's umbilical vein.In brief, process the cell of peeling off from blood vessel wall with collagenase, put into the tissue culture flasks applying with gelatin, in the M199 culture medium of Hepar Sus domestica element, 20 ug/ml endothelial cell growth supplement (ECGS) and the glutamine of the hyclone that contains 10% low endotoxin, 90 ug/ml preservative frees, cultivate.

Can be according to the people's such as Asahara method (for the separation of the endothelial progenitor cells of inferring of angiogenesis, Isolation of putative progenitor endothelial cells for angiogenesis, Science.275:964-967,1997, include in herein as a reference) from human peripheral, separate and obtain endothelial progenitor cells (EPC).By be coated with anti-CD34 antibody magnetic bead and together with the human peripheral liquid of component separation, hatch.After hatching, the cell of elution of bound, is incubated in EBM-2 culture medium (Clonetics, San Diego, CA).Or, can adopt enrichment medium partition method to separate these cells.In brief, obtain peripheric venous blood from volunteer, adopt density－gradient centrifuga－tion method to separate and obtain mononuclear cell, these cells are seeded in to (Becton Dickinson) on the cultivation slide that is coated with fibronectin, EC basal medium-2 (EBM-2) that supplement 5% hyclone, people VEGF-A, hFGF-2, human epidermal growth factor, insulin-like growth factor-i and ascorbic acid (Clonetics) in cultivation.Make endothelial progenitor cells growth 7 days, within every 48 hours, change culture medium.Fluorescent antibody by anti-CD45, CD34, CD31, VEGFR-2, Tie-2 and E-Selectin is carried out cell qualification.

In another embodiment, can adopt conventional method, mammalian cell is carried out to transfection with any expression cassette that contains any following clone gene, described clone gene can contain and is coded in uncommon specific marker molecule in circulating cells (for example prostate specific antigen or osteocyte antigen), and also can expression of peptides and/or protein, such as platelet derived growth factor (PDGF), fibroblast growth factor (FGF) or nitricoxide synthase (NOS) etc.(referring to, for example, purchased from Stratagene, San Diego, the mammalian expression vector of CA and transfection reagent box).For example, according to the method for Rosengart etc., adopt the adenovirus expression carrier of VEGF expression cDNA, with the pig endothelial progenitor cells of VEGF (VEGF) transfection purification (" be used for the treatment of and give the assessments in six months that I phase of the angiogenesis gene therapy of the adenovirus vector of VEGF expression 121 cDNA tests in the direct cardiac muscle of coronary heart disease ", 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, include in herein as a reference).

Antibody

The monoclonal antibody (" secretion pre-determines the continuous breeding method of the fused cell of specific antibody " that can adopt in the method for the invention according to the standard technique preparation of Kohler and Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 265:495-497,1975, include in herein as a reference), or can directly obtain the monoclonal antibody for the inventive method from commercially available source.Endotheliocyte can be used as the original monoclonal antibody that produces anti-surface endothelial cell antigens of immunity.

HUVEC or purified endothelial progenitor cells can be injected into the monoclonal antibody that mice or rat are prepared anti-endotheliocyte.After the sufficiently long time, put to death mice, get its splenocyte.Make immortality cell by splenocyte being blended in to myeloma cell or lymphoma cell, merge conventionally and for example, carry out containing in nonionic surfactant (Polyethylene Glycol).Make gained comprise that the cell of amalgamation hybridoma for example, grows in selective medium (HAT-culture medium), and use limiting dilution condition that the cell of survival is grown in this culture medium.Growth of Cells for example,, in suitable container (microtiter well), has required specificity, the monoclonal antibody that can react with endotheliocyte antigen in screening supernatant.

The existing multiple technology that can be used for improving monoclonal antibody productive rate, for example: hybridoma is injected into the mammalian hosts intraperitoneal of accepting these cells, then gathers in the crops ascites.When can not collect enough monoclonal antibodies from ascites time, can from this host's blood, gather in the crops antibody.Thereby existing multiple separation and the monoclonal antibody purification of can be used for makes in monoclonal antibody the not method containing other oroteins or pollutant.

Scope of the present invention also comprises the useful binding fragment of antibody (for example anti-endothelial cell monoclonal antibody), Fab, the F of the monoclonal antibody of for example these anti-endotheliocytes (ab ') ₂.Can obtain described antibody fragment by routine techniques.For example, can be prepared with by carrying out peptide enzymic digestion with papain or pepsin antagonist the binding fragment of use.These fragments can be used alone, or combine use with antibody and the fragment thereof of its origin antibody or other type.

Antibody of the present invention can be mouse IgG antibody-like; But this does not become restriction.Specific antibody, example antibody described above is with those antibody suitable with its function, no matter be mouse, mammal (comprising humanized) or other sources, or their combination, all be included in category of the present invention, also comprise other antibody-like containing isotype, such as IgM, IgA, IgE etc.These antibody capable specific recognition and with high-affinity in conjunction with the target antigen on target cell membrane, no matter be that this target antigen is on natural molecule or on the antigen through genetically engineered.With regard to antibody, term " in function quite " refers to two kinds of different antibody same antigen site on conjugated antigen separately, and in other words, described antibody competition is in conjunction with identical antigen.Described antigen can be positioned on identical or different molecule.

In one embodiment, can adopt monoclonal antibody and/or its fragment that can for example, react with surface endothelial cell antigens (, CD34).The anti-CD34 monoclonal antibody that has proved to be attached on solid phase carrier can be caught the endothelial progenitor cells in human peripheral.After catching, these CFU-GM can be divided into endotheliocyte.(Asahara etc., 1997, " for the separation of the endothelial progenitor cells of inferring of angiogenesis ", Isolation of putative progenitor endothelial cells for angiogenesis, Science 275:964-967.) hybridoma produce anti-CD34 monoclonal antibody can available from typical organization of the U.S. collect center (American Type Tissue Collection, Rockville, MD).In another embodiment, used the monoclonal antibody that can for example, react with surface endothelial cell antigens (VEGFR-1 and VEGFR-2, CD133 or Tie-2).In the embodiment of cell that uses hereditary change, similar mode available and mentioned above, adopt standard technique to produce the anti-described antibody through genetically engineered gene outcome, after substrate, be applied on the surface that described medical treatment device contacts with blood applying.

Also can use can with separate from the polyclonal antibody of accepting the homozoic anti-endotheliocyte phase reaction that this medical treatment device implants.

Support

Term " support " in this article refers in the time of insertion or implantable intravascular inner chamber, can expand any medical treatment device of intravascular space cross section.Term " support " comprising: the stainless steel or other alloy commercial stents processed that have applied by method of the present invention; Through the support covering, for example, cover with PTFE or ePTFE.In one embodiment, comprise that dermal delivery treatment coronary occlusion or sealing spleen, carotid artery, bone portion are with Tui popliteal portion blood vessel otch or aneurysmal support.In another embodiment, described stent delivery is entered to vein blood vessel.Described support can be made up of polymer or hardware, is coated with for example, substrate containing antibody and compound (somatomedin) on it.For example, can adopt deformable wire support, as authorize disclosed support in the United States Patent (USP) 4,886,062 of Wiktor, this patent is included in herein as a reference in full with it.Also can use elastic polymeric material Self-expanded stent (self-expanding stent), for example name is called " inner chamber medicament elution prothesis ", the United States Patent (USP) 5 of Intraluminal Drug Eluting Prosthesis, 871,535 and disclosed International Patent Application WO 91/12779 in the support that discloses, these patents are included in herein as a reference in full with it.Can use United States Patent (USP) 6,432, other support disclosing in 132 and 6,821,292, includes these patents herein in as a reference in full with it.Also can use following material manufacture support: rustless steel, polymer, Ni-Ti, tantalum, gold, platinum-iridium, cobalt-base alloys or Elgiloy or MP35N and other iron-bearing materials.To prop up to be placed on conduit and be delivered to therapentic part by body cavity, at this position, support be discharged from conduit, and make described support expansion for direct contact blood vessel.In another embodiment, described support comprises Biodegradable scaffold (H.Tamai, coronary stent handbook, Handbook_of_Coronary_stents, 297 pages, the 3rd edition, PW Serruys and MJBKutryk, Martin Dunitz (2000).Skilled in the art will recognize that and antibody of the present invention, somatomedin and substrate for example, can be used together with other Self-expanded stent design thing (elastic metal rack design thing).

Synthetic graft

Term " synthetic graft " refers to that anyone with Biocompatibility produces and repairs complex.In one embodiment, described synthetic graft can use polyethylene terephthalate ( pET) or politef (

ePTFE) preparation.In another embodiment, described synthetic graft is made up of polyurethane, crosslinked PVA hydrogel and/or the biocompatible foam body of hydrogel.In another embodiment, synthetic graft is by netted Merlon urethanes (polycarbonate urethane) internal layer and the outer formation of netted polyethylene terephthalate.Those skilled in the art will know that and can for example, together with graft synthetic with any biocompatibility covering component of the present invention (antibody, somatomedin and substrate), use.(Bos etc., 1998, small-bore vascular repair material: present situation, Small-Diameter Vascular Prostheses:Current Status, Archives Physio Biochem.106:100-115, includes in herein as a reference).Synthetic graft can be used for, for example, in end-end, end to side, side-end, side-side or the tube chamber of blood vessel and anastomosis of blood vessel, or for the shunting of diseased vessel section, for example, as abdominal aortic aneurysm device.

Substrate

(A) synthetic material---describedly can be selected from synthetic material for the substrate that covers support or synthetic graft, for example: biocompatible foam body or hydrophilic glucosan, for example Sensor Chip CM 5 of polyurethane, block polyurethane-urea/heparin, Poly-L-lactide, cellulose esters, Polyethylene Glycol, crosslinked PVA hydrogel, hydrogel.

(B) naturally occurring material---described substrate can be selected from naturally occurring material, for example: collagen, fibronectin, vitronectin, elastin laminin, laminin, heparin, fibrin, cellulose and carbon.To there is enough elasticity and pliability not rupture on the contact surface of support or synthetic graft to ensure it for the basic demand of substrate.

(C) fullerene---described substrate also can comprise fullerene (term " fullerene " comprises multiple fullerene molecule).Fullerene is carbon-cage (carbon-cage) molecule.The excursion of carbon (C) molecular number in fullerene is C ₂₀-C ₁₅₀.Fullerene is according to method well known to those skilled in the art, by high temperature making carbon or carbon containing class substance reaction produce; For example,, by carbon being carried out to laser vaporization, heat carbon or burn hydrocarbons in electric arc in carbon black generation flame.(authorize the United States Patent (USP) 5,292,813 of Patel etc.; Authorize the United States Patent (USP) 5,558,903 of Bhushan etc., it discloses with it and includes in herein as a reference in full).In all cases, deposit or the flue dust of carbon containing have all been produced.Can, from this flue dust, for example, extract and obtain different fullerenes by the solvent with suitable (toluene).With known method separation fullerene, especially by high performance liquid chromatography (HPLC).Can synthesize or from Dynamic Enterpri ses, Ltd. (Berkshire, England or Southern Chemical Group, LLC, Tucker, Georgia) or Bucky USA (Houston Texas) buy fullerene.

Available diverse ways deposits fullerene from the teeth outwards, described method comprises: distillation, laser vaporization, sputter, ion beam, spraying, dip-coating, roller coating or brushing are (as United States Patent (USP) 5,558, in 903, disclose, include in herein as a reference in full with it), or by the derivatization of rack surface.

A key character of fullerene is that they can form " activated carbon ".The electronic structure of fullerene is overlapping π rail system, thereby multiple bonding electrons is present in the surface around molecule jointly.(Chemical and Engineering News, Apr.8,, includes in herein as a reference by 1991, the 59 pages).As the form of the activated carbon of tool, fullerene demonstrates the basic Van der Waals force to weak interaction.The absorption property on fullerene surface makes it have the extra modification of guiding specific cell membrane interaction.For example, can for example, be adsorbed onto fullerene surface in the specific molecular of the chemical property of the cell membrane of particular cell types or specific cell membrane component (agglutinin or antibody) by thering is selective binding.Can (for example operate to produce the different cell types of selective binding to the adhesion of fullerene surface different molecular, endothelial progenitor cells, endotheliocyte, fibroblast, primary explant or T-cell subsets) surface, for example authorize the United States Patent (USP) 5 of Richmond etc., 310,669, it discloses with it and includes in herein as a reference in full; Stephen R.Wilson, the biological characteristics of fullerene, Biological Aspects of Fullerenes, Fullerenes:Chemistry, Physics and Technology, the volumes such as Kadish, John Wiley & Sons, NY 2000, includes in herein as a reference.

Fullerene also can form the nanotube that can mix other atom or molecule.(its announcement is included in herein as a reference for Liu etc., Science 280:1253-1256 (1998)).Synthetic and the preparation method of CNT is well-known in the art.(authorize the United States Patent (USP) 5,753,088 of Olk etc. and authorize the United States Patent (USP) 5,641,466 of Ebbsen etc., both announcements are all included in herein as a reference in full with it).Also can the inner side of CNT will be mixed such as protein-based molecule.For example, cutting behind nanotube two ends available enzyme (for example Zn ₂cd ₂-metallothionein, cytochrome C and C3 and beta-lactamase) filled with nanotubes.(Davis etc., Inorganica Chim.Acta 272:261 (1998); The Full Sci.Tech.5 (4) such as Cook: 695 (1997), both all include in herein as a reference).

Also can use three-dimensional fullerene structure body.Include in full herein the United States Patent (USP) 5 of authorizing Mirkin etc. as a reference in, in 338,571, disclose the 3-dimensional multi-layered fullerene structure body forming by the following method on substrate surface: (i) fullerene is carried out to chemical modification and form material (bond-forming species) to produce key; (ii) chemical treatment is carried out to provide key to form material in the surface of base material, described material be enough to solution in there is key and form the fullerene covalent bond of material; And (iii) modified fullerene solution is contacted with treated substrate surface, to form the Fullerene layer that is covalently bonded in treated substrate surface.

(D) substrate is put on to medical treatment device

Described substrate should be able to be attached to the medical treatment device surface of (comprising support or synthetic graft) securely.In one embodiment, this is to realize by applying continuous thin-layer matrix.Or, can only antibody and somatomedin be applied on the outer surface directly contacting with intravascular space.Apply serially dissimilar substrate pantostrat.After substrate is applied on support, can be by described antibody covalently or non-covalently in conjunction with being coated in substrate.

In order to cover medical treatment device, for example support, available moderately viscous matrix liquid solution impregnation or spray described support.Apply after each layer, before applying lower one deck, made support dry.In one embodiment, the gross thickness of paint sample matrix coating is no more than 100 microns.

In one embodiment, the rack surface of hypothallus, for for example, first through functionalization, has then been added in the surface of described medical treatment device.,, antibody and other coating component (for example somatomedin) are coupled to stromal surface thereafter.At this on the one hand, substrate is applied over such as the technology of rack surface and can produces functional chemical group.For example, described chemical group can be amino, and it can be with the functional group reactions of polymer for example to fix, as the substrate intermediate layer of the carrier of identification and the part (antibody, peptide and/or somatomedin) of acquisition target cell.

In another embodiment, can prepare in the following way suitable substrate coating solution: under aseptic condition, by 480 milligrams of (mg) pharmaceutical carriers, for example poly--D, L-lactide is (as R203 purchased from Boehringer Inc., Ingelheim, Germany) be dissolved in 3 milliliters of (ml) chloroform.But, can use in principle blood-and biodegradability (or the abiotic degradability) substrate of the tissue-compatibility (biocompatibility) and solubilized, dispersion or emulsifying, as long as can be dried quickly after using as certainly adhering to paint sample or coating sample coating on medical treatment device.

For example, those of ordinary skill in the art knows usable fibers albumen drug delivery medical device.Disclose by Fibrinogen is contacted with thrombin and can make fibrin condense authorizing in the United States Patent (USP) 4,548,736 of Muller etc. (it discloses in full and includes in herein as a reference).Preferably, fibrin in fibre-bearing albumen support of the present invention contains the XIII factor, and in condensing, there is calcium, (as authorize institute in the United States Patent (USP) 3,523,807 of Gerendas and disclose, its announcement is included in herein as a reference in full, or disclose as disclosed european patent application 0366564, its announcement is included in herein as a reference in full), to improve mechanical performance and the biological stability of implanting device.In this embodiment, for example, for fear of any interspecies immunity reaction (the anti-cattle of people), derive from and the animal or human of animal or human's identical type that will implant this support for the preparation of the fibrinous Fibrinogen of the present invention and thrombin.The fibrin product of producd fibers extract of protein fine film form in the following manner: the Fibrinogen of mixing and thrombin are poured into thin film, then remove moisture by semipermeable membrane diafiltration from described thin film.In european patent application 0366564, (its announcement is included in herein as a reference in full) disclosed base material (preferably have highly porous or thrombin or Fibrinogen are had to high-affinity) contacted with thrombin solution with fibrinogen solution.Result is by fibrinogenic polymerization, forms fibrin layer on the surface of medical treatment device.The multi-layer fiber albumen applying by the method can provide the fibrin layer of any desired thickness.Or, can first make fibrin condense, be then ground into powder, will in this powder and the mixed mould that is incorporated in heating of water, be embossed into required form (United States Patent (USP) 3,523,807).Can be by fibrin for example, contact with fixative (glutaraldehyde or formaldehyde), the fibrin of generation shaping and obtain the stability of raising.Can be by well known by persons skilled in the art for the preparation of being used for the present invention with fibrinous these methods of formation and other method.

If apply described synthetic graft with collagen, prepare collagen and the method that forms coating on synthetic graft device is known, as authorize the United States Patent (USP) 5,851 of Weadock etc., in 230, disclose, its announcement is included in herein as a reference in full.This patent has been described the method for apply synthetic graft with collagen.Generally include collagen dispersion liquid is applied over to base material for the method that collagen is adhered to porous graft base material, it is dried and repeats this process.The preparation of collagen dispersion is normally by being mixed into insoluble collagen (about 1-2 % by weight) dispersion liquid of acid pH (pH is 2-4).Conventionally described dispersion liquid is entered in graft inner chamber with injector to inject, and with hands rub press make collagen serosity cover whole inner surface area.Remove excessive collagen serosity from one of two openings of described graft.Repetitive coatings and drying steps are for several times to provide enough processing.

In another embodiment, with amorphous carbon drug delivery medical device or synthetic graft.At United States Patent (USP) 5,198, in 263 (its announcement is included in herein as a reference in full) described a kind of under fluorinated gas or the existence containing the gas of other halogen, the method for producing height ratio low temperature depositing amorphous c film.The deposition of carrying out according to the method for this invention can (comprise ambient room temperature) at the temperature lower than 100 DEG C, and, chemical vapor deposition process auxiliary with radio frequency, plasma carries out.The amorphous c film of using method generation of the present invention can adhere to the base material of numerous species well, and described base material for example comprises: glass, metal, quasiconductor and plastics.

Fullerene molecule is combined with the reactive amino group site of amido polymer with form fullerene-support, containing amino polymer can be by United States Patent (USP) 5,292,813 (its announcement is included in herein as a reference in full) complete.The chemical modification of being undertaken by this mode can make fullerene directly mix in support.In another embodiment, can as described above fullerene be deposited on the surface of support or synthetic graft.(referring to, authorize the WO 99/32184 of Leone etc., its announcement is included in herein as a reference in full).Fullerene (for example C ₆₀) also can be attached to (Yamago etc. on stainless steel surfaces by epoxy bond, " Organic fullerene forms with C-C bonding react chemically derived by oxidation, reduction and C-O ", Chemical Derivatization of Organo fullerenes through Oxidation, Reduction and C-O and C-C Bond Forming Reactions, J.Org.Chem., 58 4796-4798 (1998), its announcement is included in herein as a reference in full).This adheres to is by completing with the covalent bond of oxygen.Described compound and coupling scheme can be buied from BuckyUSA. (BuckyUSA, Houston, Texas).

(E) part (for example antibody, peptide and/or somatomedin) is added to substrate---can promote the antibody of endothelial progenitor cells adhesion and the somatomedin of energy Promote cell's growth and differentiation to be covalently or non-covalently incorporated in substrate.Can be in the following way for example, by part coating (antibody, antibody fragment, hormone, peptide, somatomedin and/or like that) doped matrix layer: part is mixed with matrix coating solution, then described solution is applied on the surface of device.In some embodiments, antibody, fragment or their combination and/or somatomedin are attached to the outermost surface of the substrate that is applied over device surface of internal cavity, thereby make part (for example antibody) give prominence on this surface and can contact with blood circulation, and keep their affinitys to target cell.In these embodiments, can adopt standard technique that described part (for example antibody) is applied in stromal surface.

In one embodiment, described antibody is added in the solution containing substrate.For example, with the concentration of 500-800mg/dl, the Fab fragment of anti-CD34 monoclonal antibody and the solution that contains human fibrinogen are hatched jointly.The concentration that should understand anti-CD34 Fab fragment can be different, and those of ordinary skill in the art just can determine optium concentration without the test through too much.Support is added in Fab/ fibrin mixture, then add concentrated thrombin (concentration is at least 1000U/ml) to carry out activated fiber albumen.The polymer fiber mixed liquid of protein that gained is contained to Fab fragment directly adds substrate, on the surface of support or synthetic graft, is pressed into thin film (being thinner than 100pm).In fact, before drug delivery medical device or synthetic graft, in this way can be by the antibody of any type or antibody fragment doped matrix solution.

For example, in another embodiment, the whole antibody of with or without antibody fragment and somatomedin are that covalent coupling is in substrate.In one embodiment, by heterogeneous or homogeneity bifunctional linker molecule, described antibody and one or more somatomedin are covalently attached to substrate.As used herein, term " connect (tethered) " refers to by linkers antibody covalent coupling in substrate.Adopt using of linkers related to the present invention to be usually directed to before substrate is attached to support, by linkers covalent coupling in described substrate.At covalent coupling, after substrate, linkers provides many functional activity groups that can be used for one or more antibody of covalent coupling for substrate.In an embodiment of this embodiment, Figure 1A provides by the example of corsslinking molecular coupling.Endotheliocyte 1.01 is incorporated into antibody 1.03 by surface antigen 1.02.Described antibody is connected in substrate 1.05-1.06 by corsslinking molecular 1.04.Substrate 1.05-1.06 is attached to support 1.07.Described linkers can directly be connected in substrate and (that is, pass through carboxyl), or by the coupled chemical reactions of knowing, for example esterification, amidation and acylation.Described linkers can be two or triamido functional compound, and they are connected with substrate by direct formation amido link, and provide can with the amido functional group of antibody response.For example, described linkers can be polyamines functional polymer, as polymine (PEI), polyene propyl amides (PALLA) or Polyethylene Glycol (PEG).Can be by Shearwater Corporation (Birmingham, Alabama) buy the scheme of multiple polyethyleneglycol derivative (for example, mPEG-succinyl imidodicarbonic diamide base propionic ester or mPEG-N-N-Hydroxysuccinimide) and covalent coupling.(also can be referring to, Weiner etc., Polyethylene Glycol sept is for passing through the impact of immobilized antibody capture antigen, Influence of a poly-ethyleneglycol spacer on antigen capture by immobilized antibody, J.Biochem.Biophys.Methods 45:211-219 (2000), includes in herein as a reference).Be understood that the selection of concrete coupling agent can be depending on the type of antibody used, and this type of is selected without making through too much examination.Also can use the mixture of these polymer.These molecules contain multiple drapability amido functional group, and these functional groups can be used for surface and fix one or more antibody, peptide, protein, hormone and other coating composition.

In one embodiment, antibody can be attached to the C of Direct precipitation on rack surface ₆₀fullerene layer.Cross-linking agent can be covalently attached to fullerene.Then make antibody be attached to cross-linking agent, and then adhere to support.Figure 1B provides by fullerene C ₆₀the example of coupling.Endotheliocyte 2.01 is incorporated into antibody 2.03 by cellular antigens 2.02, and then is covalently or non-covalently incorporated into substrate 2.04.Substrate 2.04 is passed through C ₆₀2.05 covalent couplings are in support 2.06.

Micromolecule of the present invention can comprise synthetic or naturally occurring molecule or the peptide for replacing antibody, antibody fragment, somatomedin etc.For example, agglutinin is a kind of naturally occurring nonimmune source property sugar binding peptide.Agglutinin antigen (Ulex Europaeus the Uea 1) (Schatz etc. 2000 of endothelial cell specific, people's endometrium endotheliocyte: tissue factor separates with 1 type blood plasminogen activator inhibitor, characterized and inflammation mediated expression, Human Endometrial Endothelial cells:Isolation, Characterization, and Inflammatory-Mediated Expression of Tissue Factor and Type 1 Plasminogen Activator Inhibitor., Biol Reprod 62:691-697), the for example alternative cell surface that is incorporated into endothelial progenitor cells.

Create synthetic " micromolecule " of the different cell surfaces of energy targeting, protein, glycoprotein, polysaccharide and receptor.The specific surface molecular of these molecular energy selective binding, and the specific cell type of energy targeting, for example endothelial progenitor cells.Synthetic micromolecule can be identified endothelial cell surface labelling, for example VEGF.SU11248 (Sugen Inc.) (Mendel etc., 2003, " anti-tumor activity in vivo of a kind of Novel tyrosine kinase inhibitors SU11248 taking vascular endothelial growth factor receptor and platelet derived growth factor receptor as target: the mensuration of pharmacokinetics/pharmacodynamics mutual relation ", In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors:determination of a pharmacokinetic/pharmacodynamic relationship, Clin Cancer Res.Jan, 9 (1): 327-37), PTK787/ZK222584 (Drevs J. etc., 2003, " receptor tyrosine kinase: some new main targets of anticancer treatment ", Receptor tyrosine kinases:the main targets for new anticancer therapy, Curr Drug Targets.Feb, 4 (2): 113-21) and SU6668 (Laird, AD etc., 2002, " in vivo SU6668 to the inhibitory action of Flk-1/KDR and PDGFR-β causing the rapid apoptosis of tumor vessel system in Mice Body and tumor decline ", SU6668 inhibitsFlk-1/KDR and PDGFRbeta in vivo, resulting in rapid apoptosis of tumor vasculature and tumor regression in mice.FASEB J.May, 16 (7): 681-90) be the micromolecule in conjunction with VEGFR-2.

Another subclass synthesized micromolecule of targeting endothelial cell surface is α (v) β (3) integrin inhibitors.SM256 and SD983 (Kerr JS. etc., 1999, " new small molecule material α v integrin antagonist: compare with anticancer effect of known various angiogenesis hormone class mortifiers ", Novel small molecule alpha v intergrin antagonists:comparative anti-cancer efficacy with known angiogenesis inhibitors.Anticancer Res Mar-Apr; 19 (2A): 959-68) being can targeting and the synthesized micromolecule that is incorporated into α (v) β (3) on endothelial cell surface.

The invention provides a kind of drug delivery system, it comprises: the medical treatment device of coating, for example support, stent graft, cardiac valve, conduit, vascular repair screen, artificial heart, external and built-in left ventricular assist device (LVADs) and synthetic blood vessel graft, they can be used for treating disease, these diseases comprise: tumor and angiopathy, for example: restenosis, atherosclerosis, thrombosis, angiemphraxis etc.In one embodiment, the coating on medical treatment device of the present invention comprises: the substrate of biocompatibility; At least one antibody, antibody fragment or their combination; And/or at least one compound, as part or therapeutic agent (as estradiol, angiogenesis factor, FGF etc.).

In one embodiment, transgenic cell is mixed with at least one metastatic gene, and described metastatic gene can be by taking virus or non-virally import described cell as basic genetic method.Described at least one medicine of metastatic gene codified, can continuous expression or express under the induction stimulating.In one embodiment, described medicine can be hormone, peptide, protein etc.On the surface of described transgenic cell, also have at least one antigen, described antigen can be coated on the antibody of surfaces of medical devices and identify and combination.

As used herein, " antibody " refers to antibody or antibody fragment, or the combination of antibody and fragment, and they can be monoclonal antibody, polyclonal antibody, chimeric antibody or humanized antibody.Antibody fragment of the present invention comprises the antibody fragment (for example macromole and micromolecule) of any size, and these fragments retain the identification identical with this antibody and the characteristic (Figure 1A, 1B and 11) in conjunction with target antigen.

As used herein, " part " refers to can for example, in conjunction with the molecule of another kind of molecule (receptor on mammalian cell).For example, part can be antibody, antibody fragment (Figure 1A, 1B, 11 and 17), cell adhesion molecule or basement membrane composition, and they can be identified and in conjunction with specificity epitope on target cell membrane or structure.Adopting in the embodiment of mammalian cell of hereditary change, can identify through special selection for the part in medical treatment device coating and in conjunction with the gene outcome being produced by the foreign DNA importing in transgenic cell.

As used herein, " protein " refers to the amino acid polymer of any length.Described polymer can be straight or branched, can comprise modified aminoacid, also can between be inserted with non-aminoacid.Described polymer can be naturally occurring peptide, protein, or its modification and synthesized form, comprises it: bioactive fragment, derivant, congener, analogies and nonfunctional or dominant negative mutant.

Described medical treatment device can be any device for the organ in implantation belt chamber or body area, it can include but not limited to: support, stent graft, synthetic blood vessel graft, cardiac valve, conduit, vascular repair screen, pacemaker, pacemaker lead, defibrillator, in hole, ovum garden patent (PFO) every closing device, vascular clamp, vascular aneurysms dead lock, hemodialysis graft, hemodialysis catheter, chamber diverter, aortic blood tuberculation graft device or assembly, venous valve, suture, blood vessel anastomosis clamp, remain-type vein or ductus arteriosus, vagina vasorum and drug conveying mouth.According to the difference of device, described device can be made from a variety of materials.For example, support of the present invention can be made with rustless steel, Nitinol (NiTi) or evanohm.Synthetic blood vessel graft can be made by crosslinked PVA hydrogel, politef (PTFE), expanded polytetrafluoroethyltoe (ePTFE), porous high density polyethylene (HDPE) (HDPE), polyurethane and poly terephthalic acid vinyl acetate.

The biocompatible matrix that forms apparatus of the present invention coating comprises: synthetic material, for example polyurethane, block polyurethane-urea/heparin, Poly-L-lactide, cellulose esters, Polyethylene Glycol, polyvinyl acetate, glucosan and gelatin; Naturally occurring material, for example basement membrane components, as collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose; And amorphous carbon or fullerene etc.

In one embodiment, described medical treatment device comprises the biocompatible matrix that contains fullerene.In this embodiment, the carbon number of described fullerene can be about C ₂₀-C ₁₅₀, more particularly, described fullerene is C ₆₀-C ₇₀.Fullerene of the present invention also can be arranged as nanotube in surfaces of medical devices.

The antibody that offers described medical treatment device coating comprises that at least one can be identified and in conjunction with the antibody of transgenic cell surface antigen, this antigen can be expressed and can be regulated the adhesion of described cell to surfaces of medical devices by exogenous gene or metastatic gene.Described antibody can be covalently or non-covalently attached in stromal surface, or is covalently attached to the outermost layer of the substrate that covers medical treatment device by linkers.Of the present invention this on the one hand, for example, described monoclonal antibody also can comprise Fab or F (ab ') ₂fragment.

Described antibody can be identified and the subject mammiferous antigen of combination docking specifically, and their specificity does not depend on cell line.In one embodiment, described antibody on human endothelial progenitor cells surface antigen has specificity, and described surface antigen is for for example: CD133, CD14, CD34, CDw90, CD117, HLA-DR, VEGFR-1, VEGFR-2, Muc-18 (CD146), CD130, stem cell antigen (Sca-1), stem cell factor 1 (SCF/c-Kit part), Tie-2, HAD-DR and other antigen (for example anti-H-2K ^kantibody).

In another embodiment, the coating of described medical treatment device comprises at least one deck biocompatible matrix as above, and this substrate comprises at least one micromolecular outer surface natural or synthetic source for the treatment of effective dose for adhering to.Described micromolecule can identify transgenic cell surface antigen and with its interaction, thereby transgenic cell is fixed on the surface of device, and induce the expression of metastatic gene.Described micromolecule can, from various sources, for example, from cellular component, as fatty acid, protein, nucleic acid, saccharide etc., and can interact with transgenic cell surface receptor.In this embodiment of the present invention, the coating on medical treatment device also can comprise compound, the part of being for example combined with the coating that contains antibody.

Taking virus and non-viral as basic genetic method all can be used for importing metastatic gene with generation transgenic cell.The medicine by the of short duration or stable metastatic gene coding mixing can be expressed and secrete to transgenic cell of the present invention.Also can be in conjunction with other metastatic gene to give its survival, selectivity and/or growth vigor.Can modify to produce transgenic cell non-renewable or that breed again to various cells, these cells are for for example: endotheliocyte or granulocyte, comprise neutrophil cell, eosinophile granulocyte, basophilic granulocyte, mononuclear cell and lymphocyte or somatic cell, or the combination of these cells.Can cultivate in vitro transgenic cell, it is carried out to Collection and preservation.Can generate the transgenic cell that can produce various medicines by mixing different metastatic genes, for different therapeutic purposes.Can pass through whole body or local approach, give transgenic cell with single or mixed cellularity group.For individual condition of illness, can give not commensurability transgenic cell with the not commensurability medicine of secretion.In one embodiment, the renewable endothelial progenitor cells of described transgenic cell.In another embodiment, can use the mode of sending based on conduit or give transgenic endothelial progenitor cells with two airbag inflation methods (dual balloon inflation method) part.

In one embodiment, transgenic cell also comprises other metastatic gene of expressing exogenous cell surface antigen, and these antigens can be coated on described antibody specificity in medical treatment device substrate and identify and combination.The expression of metastatic gene and the secretion of product can be to be recurred or activates inducibility promoter and temporarily occur at exogenous stimulation.

The therapeutic compound of being encoded by metastatic gene of the present invention can be any molecule with required physiological action, it can be but is not limited to: defined protein-based, comprise that somatomedin, chemotactic factor and cytokine, part and receptor and other functional protein and nonprotein can secrete compound.In one embodiment, therapeutic compound is to be selected from the protein of lower group: endothelial nitric oxide synthase (eNOS), VEGF (VEGF), the anti-inflammatory factor and inflammation regulatory factor.

Medicine, for example, in the embodiment of mammalian cell that adopts hereditary change, to stimulate the compound that metastatic gene is expressed and target product is secreted, can be part or other composition in described medical treatment device coating, they can and activate the downstream signal pipeline of extrachromosomal nucleic acid (for example importing the DNA construction in target cell) in conjunction with transgenic cell surface antigen.In another embodiment, the metastatic gene of the mammalian cell of hereditary change is expressed and can be activated by for example part or medicine, and described part or medicine can be taken in by described transgenic cell and express by inducibility promoter stimulated gene.In one embodiment, described part or medicine are general administrations.In another embodiment, described part or medicine are to be coated in the substrate of implanting device and topical.

The invention provides the method that is used for the treatment of various diseases, described disease can be but is not limited to: tumor, angiopathy and healing reaction.Described method relatively and the progress of prior art be as required various medicines are carried out to target site send aequum.

The invention provides a kind of method for the treatment of tumor and transfer thereof.In this embodiment, described metastatic gene codified (1) anti-angiogenesis, for example interferon (IFN), thrombospondin (TSP), angiostatin, Endostatin, oncostatin M (OSM) and Rho, they can suppress to generate as the new vessels of carrying out property of tumor growth precondition; Or (2) tumor suppressor protein, the kinases (CDK) that the antibody of for example p53, Rb, E1, BRCA1, Growth of Cells activator (for example somatomedin) or dominant negative mutant or cyclin rely on or cyclin, E2F, NFKB; Or the combination of these genes.In one embodiment, described metastatic gene can comprise the gene of the following material of encoding: for example, and prostacyclin and/or cyclooxygenase, α-CGRP, matrix metalloprotease and/or endothelial nitric oxide synthase.

As used herein, phrase " anti-angiogenesis " refers to the molecule that can suppress angiogenesis or angiogenic growth.

The present invention also provides the method that is used for the treatment of angiopathy.In one embodiment, a kind of method for the treatment of ischemic disease is provided, metastatic gene is used for the angiogenesis factor of encoding in the method, for example antibody or the dominant negative mutant of the multiple-effect factor (pleiotrophin), angiogenesis factor, angiogenin, integrin stimulating factor and/or anti-angiogenesis.

As used herein, phrase " angiogenesis factor " refers to the molecule that can stimulate angiogenesis or angiogenic growth.

In another embodiment, described invention is used for the treatment of atherosclerosis, restenosis, thrombosis, aneurysm or angiemphraxis.In this embodiment of the present invention, metastatic gene promotes for coding (a) eNOS or the VEGF that endothelium is rebuild; Or (b) anti-inflammatory or inflammation regulatory factor, for example IFN-β, IFN-α, TGF-β or interleukin 10 (IL-10); Or (c) for suppressing smooth muscle cell growth, migration or the differentiation inhibitors of neointimal hyperplasia; Or the combination of these genes.

The present invention also provides the engineered method for inducing healing reaction.In one embodiment, provide a kind of and merged at the implanting device surface of internal cavity rapid induction being placed in implantable intravascular target lesion the method that endodermis forms, transgenic cell is wherein the endothelial progenitor cells of expressing eNOS, VEGF or the anti-inflammatory factor or inflammation regulatory factor.In this embodiment, compared with prior-art devices, the biocompatibility of medical treatment device of the present invention increases, can be deposited on the implant site of medical treatment device along surface of internal cavity by reducing or suppressing smooth muscle cell migration, smooth muscle cell differentiation and collagen, and reduce or excessive neointimal hyperplasia and the restenosis of inhibition based on tissue.

In one embodiment, comprise the following steps for the method for coated medical devices: the surface that one deck biocompatible matrix is at least applied over to medical treatment device, wherein said biocompatible matrix can comprise at least one component that is selected from lower group: polyurethane, block polyurethane-urea/heparin, Poly-L-lactide, cellulose esters, Polyethylene Glycol, polyvinyl acetate, polysaccharide (for example glucosan, gelatin), collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose and carbon and fullerene, and simultaneously or on described biocompatible matrix, apply successively at least one antibody and the optional a kind of compound that can induce metastatic gene to express.

The present invention also provides a kind of method that is used for the treatment of disease, and described disease is for for example, mammiferous tumor, angiopathy and wound healing.The present invention includes medical treatment device is implanted in mammiferous blood vessel or pipe, wherein said medical treatment device is coated with: (a) biocompatible matrix; (b) at least one antibody; Optionally (c) a kind of compositions; Transgenic cell is imported to the described mammal that needs described treatment, and optionally give compound, wherein be coated on the antibody recognition in medical treatment device substrate and be combined in the antigen of expressing on transgenic cell surface, thereby described transgenic cell is fixed on the surface of substrate, and at least one medicine of being encoded by metastatic gene is by the cellular expression being fixed described in being activated by compound, described compound is for example medicine and at the therapeutic gene product of specifying site secretion.

The present invention also provides a kind of method that is used for the treatment of mammal medium vessels disease, described method comprises: medical treatment device is implanted to described mammiferous blood vessel or pipe, and wherein said medical treatment device is coated with: (a) biocompatible matrix; (b) at least one antibody; Optionally (c) a kind of compositions; ; Transgenic cell is imported to the described mammal that needs described treatment, and optionally give compound, wherein be coated on the antibody recognition antigen that also combination is only expressed on transgenic cell film surface in medical treatment device substrate, thereby described transgenic cell be fixed on the surface of substrate.Described transgenic (hereditary change) cell also can comprise the hereditary material of at least one therapeutic gene product of encoding, and described gene outcome can be continuous expression or for example, expresses in the time that signal (compound including hormone and peptide) activates.

Transgenic cell of the present invention can comprise at least one effable metastatic gene, this gene can be used for coding (but being not limited to): (1) somatomedin, comprise its family member: for example platelet derived growth factor (PDGF), transforming growth factor (TGF), endothelial cell growth factor (ECGF) (EGF), fibroblast growth factor (FGF), insulin like growth factor (IGF), VEGF (VEGF), heparin-bounding somatomedin, the somatomedin (HDGF) that hepatocarcinoma is derivative, hepatocyte growth factor/dispersion factor (HGF), placental growth factor (PIGF), platelet-derived endothelial cell growth factor (ECGF) (PD-ECGF), stem cell factor (SCF), and their other albumen form, (2) chemotactic factor: for example CXC family, CC family, C family, and their other albumen form, (3) cytokine: for example adam protein (ADAM), annexin V, B7 & CD28/CTLA-4 receptor family, bone morphogenetic protein (BMP), caspase, CD44, CD44H, endothelin-1 (ET-1), eph, erythropoietin (Epo), intercellular adhesion molecule-3/CD50 (ICAM-3), macrophage stimulating protein (MSP), matrix metalloproteinase (MMP), neurotrophic factor, endothelial nitric oxide synthase (eNOS), NKG2D, PECAM-1 (PECAM-1/CD31), the multiple-effect factor/midkine (PTN/MK), TfR (sTfR), protection peptide (hedgehog peptide), STAT, stem cell labeling, Th1/Th2, thrombopoietin (Tpo), tnf family cytokines, VCAM-1/CD16, the non-specific inhibitive factor β of monoclonal (MNSF β), 6Ckine (SLC), B-lymphocyte chemotactic inducer (BCA-1/BLC), leukaemia inhibitory factor, the peptide (GRO) of the neutrophil activation of monocyte derived, and their other albumen form, (4) participate in other functional protein of signal conduction adjusting, Cycle Regulation, cell division and/or cell differentiation, they are for for example: part, receptor, phosphorylase, kinases, transcription factor, and their other albumen form.

In one embodiment, for example be for anti-angiogenesis of the present invention: the blocker of interferon (IFNs), thrombospondin (TSP), angiostatin and Endostatin, oncostatin M (OSM), integrin arrangement, inhibitors of metalloproteinase, endotheliocyte phosphorylation inhibitor, for the antibody of the dominant receptor of blood vessel generation derivant, blood vessel generation derivant, by the protein of alternate manner effect, and their other albumen form.Other angiogenesis factor, comprises angiogenesis factor, angiogenin, integrin stimulating factor (for example Del-1), and their other albumen form.

For other somatomedin of the present invention for for example: the multiple-effect factor, midkine, VEGF family (comprises VEGF-2, VEGF-C and VEGF-D), FGF family (comprises FGF-1, FGF-2, FGF-5 and FGF-18), the somatomedin (HDGF) that hepatocarcinoma is derivative, hepatocyte growth factor/dispersion factor (HGF), endothelial cell growth factor (ECGF) (EGF) family member (comprises transforming growth factor α, EGF and TGF-α-HIII and platelet derived growth factor (PDGF) (comprise AA, AB and BB hypotype).

Test example

In following tentative detailed description part, the present invention will be described.These following parts are in order to understand the present invention, instead of and should not be construed as the scope of the invention limiting by any way by described in the claims after embodiment.

Embodiment 1

The phenotype of endothelial progenitor cells

Separate by the following method endothelial progenitor cells (EPC): by CD34+ magnetic bead partition method (Dynal Biotech) or as enrichment medium partition method (the Asahara T describing recently, Murohara T, Sullivan A etc., " feeder vessels generates the separation method of the endothelial progenitor cells of inferring of use ", Isolation of putative progenitor endothelial cells for angiogenesis, Science 1997; 275:964-7).In brief, obtain peripheric venous blood from healthy male volunteers, and obtain mononuclear cell component by density gradient centrifugation, then this cell is inoculated in to (Becton Dickinson) on the cultivation slide that is coated with people's fibronectin, culture medium be EC basal medium-2 (EBM-2) (Clonetics), be wherein supplemented with 5% hyclone, people VEGF-A, hFGF-2, people's endothelial cell growth factor (ECGF), insulin-like growth factor-i and ascorbic acid.Make EPC growth 7 days, within every 48 hours, change culture medium.The results are shown in Fig. 2 A and 2B of these tests.Fig. 2 A has shown that with 2B the cell that anti-CD34 separates seems more seemingly spindle sample, and this just shows that these cells are just being divided into endotheliocyte.

Measure the phenotype of EC by immunohistochemical method.In brief, the phosphate buffered solution (PBS) (Sigma) of 2% paraformaldehyde (PFA) for EPC (Sigma) is fixed to 10 minutes, with PBS washing 3 times, and with the dyeing of different EC specific marker: rabbit human VEGFR-3 resistant-2 (Alpha Diagnostics Intl.Inc.), mouse-anti-human T ie-2 (Clone Ab33, Upstate Biotechnology), Mus antihuman CD 34 (Becton Dickinson), EC-agglutinin (Ulex Europaeus Uea1) are (Sigma) and mouse-anti people 8 factors (Sigma).Make two of cells contacting Fluorescein isothiocyanate (FITC) combination resist to verify the existence of antibody.Iodate the third ingot (PI) is used as to core labelling.These tests the results are shown in Fig. 2 C-2G.Fig. 2 C shows in culture has VEGFR-2 to express after 24 hours, this has just confirmed that described cell is endotheliocyte.Fig. 2 D and 2F are depicted as the nuclear staining of hatching rear combination cell for 7 days, and Fig. 2 E and 2G are depicted as the cell of using the anti-Tie-2 antibody staining of FITC coupling in the same visual field.

To eNOS mRNA carry out reverse transcriptase-polymerase chain reaction (rt-PCR) measure EPC express endothelial nitric oxide synthase (eNOS, a kind of EC function mark) ability.EPC is grown 7 days in EBM-2 culture medium, then use the total RNA test kit of GenElute mammal (Sigma) to separate total RNA, and quantitative by absorbance under 260nm.With 1 μ g random primer, the total RNA of 20 μ L is carried out to reverse transcription with Omniscript RT test kit (Qiagen).For each RT product, end reaction volume (2-10 μ L) to equal portions in two parallel PCR reactions increases, use eNOS Auele Specific Primer (299bp product, there is justice 5 '-TTCCGGGGATTCTGGCAGGAG-3 ', SEQ IDNO:1, antisense 5 '-GCCATGGTAACATCGCCGCAG-3 ', SEQID NO:2) or GAPDH Auele Specific Primer (343bp product, there is justice 5 '-CTCTAAGGCTGTGGGCAAGGTCAT-3 ', SEQ ID NO:3, antisense 5 '-GAGATCCACCACCCTGTTGCTGTA-3 ', SEQ ID NO:4) and Taq polymerase (Pharmacia Biotech Amersham).PCR circulation is as follows: 94 DEG C 5 minutes, 65 DEG C 45 seconds, 72 DEG C 30 seconds (being 35 circulations to eNOS, is 25 circulations to GAPDH).Analyze rt-PCR product by 2% agarose gel electrophoresis, with ethidium bromide colour developing, and optical density standard measure.This test the results are shown in Fig. 3 A and 3B.From Fig. 3 A and 3B, cell is hatched after 3 days under the condition that has or do not exist oxygen in culture medium, has expressed nitricoxide synthase (eNOS).Cultivate and after 7 days, still continue to express eNOS mRNA.The expression of eNOS mRNA shows that described cell has been divided into ripe endotheliocyte in 3 days, and starts to exercise the function that is similar to the endotheliocyte breaking up completely.

Embodiment 2

With anti-CD34 (antibody) apply rustless steel disk catch endotheliocyte: in process of the test, Human umbilical vein endothelial cells (HUVEC) (American type culture collection, American Type Culture Collection) is incubated in endothelial cell growth culture medium.Cell is contained with surface or together with the sample applying with CMDX and gelatin of binding antibody or rustless steel (SST) sample of blank, not hatching.After hatching, remove culture medium, and by PBS washing sample 2 times.Fixed cell 10 minutes in 2% paraformaldehyde (PFA), with PBS washing 3 times, each 10 minutes to guarantee to remove all fixatives.At room temperature, each sample is hatched 30 minutes together with lock solution, to seal all non-specific binding.By PBS washing sample 1 time, and 1: 100 diluent that contacts VEGFR-2 antibody is cultivated and is spent the night.Then use PBS washing sample 3 times, to guarantee to remove all primary antibodies.In each sample, add FITC coupling two anti-of doing the dilution of 1: 100 by lock solution, incubated at room 45 minutes on Belly Dancer device.After hatching, by PBS washing sample 3 times, wash once with the PBS that contains 0.1%Tween 20, then wash with PBS.With iodate the third ingot (PI) fixed sample, and observe under Laser Scanning Confocal Microscope.

Fig. 4 A-4E has applied CMDX described above and anti-CD34 antibody (Fig. 4 A), gelatin and anti-CD34 antibody (Fig. 4 B), blank SST (Fig. 4 C), CMDX coating but without antibody (Fig. 4 D) and gelatin coating but without the SST sample microphotograph of antibody (Fig. 4 E).Accompanying drawing is depicted as by PI and dyes and demonstrate the sample that only applies antibody and comprise many cells that adhere to sample surfaces.Blank SST comparing disk shows that few cell adhesion is in its surface.

Fig. 5 A-5C has applied CMDX and the microphotograph that is incorporated into its surperficial control sample without antibody.Fig. 5 A demonstration is dyeed visible few cell adhesion in the surface of sample by PI.Fig. 5 B shows that the cell adhering to is the VEGFR-2 positive, and this shows that they are endotheliocytes, and Fig. 5 C is depicted as the combination of core and the positive green fluorescence of VEGFR-2 of dyeing.Fig. 5 D-F has applied gelatin and in its surface without the microphotograph of the control sample of antibody.Fig. 5 D shows acellular existence, and this is because this sample is without the PI green fluorescence (referring to Fig. 5 E and 5F) of also not launching that dyes.

Fig. 6 A-6C is the microphotograph that has applied CMDX and had the SST sample of the anti-CD34 antibody that is incorporated into its surface.These accompanying drawing show samples comprise many adherent cell, and as shown in green fluorescence, they have been set up and approached the monolayer (Fig. 6 A) of fusion, have been VEGFR-2 positive cell (Fig. 6 B and 6C).Similarly, the microphotograph of the sample of Fig. 6 D-6F gelatin that is the coating that has anti-CD34 antibody of its surface combination.These accompanying drawings also, by the much red staining core and the green fluorescence that produce from VEGFR-2/FITC antibody, have shown that HUVEC adheres to sample surfaces (Fig. 6 E and 6F).

Embodiment 3

The VEGFR-2 of endothelial progenitor cells dyes with Tie-2: separate as described in example 1 above the CFU-GM obtaining in human blood, incubated in vitro 24 hours, 7 days and 3 weeks in culture medium.After hatching, remove growth medium, use PBS washing sample 2 times.With 2% paraformaldehyde (PFA) fixed cell 10 minutes, then, with PBS washing 3 times, each 10 minutes, guarantee to remove all fixatives.Together with the sheep (for VEGFR-2) of each sample and 440 μ l or horse (for Tie-2) confining liquid, hatch at room temperature 30 minutes, to seal all non-specific binding.By PBS washing sample 1 time, then add VEGFR-2 or the Tie-2 antibody of doing dilution in 1: 100 with confining liquid, hatch sample and spend the night.Then with PBS washing 3 times, to guarantee that all primary antibodies are all washed away.To add in each sample with horse or sheep confining liquid do the dilution of 1: 100 FITC coupling two anti-(200 μ l), and at room temperature hatch 45 minutes on Belly Dancer device.After hatching, use PBS washing sample 3 times, use the PBS that contains 0.1%Tween 20 to wash once, then with washing in PBS.With iodate the third ingot (PI) fixed sample, and observe under Laser Scanning Confocal Microscope.

The microphotograph of the sample of Fig. 7 is the coating that contains CD34 antibody on its surface CMXD, this sample has been hatched 24 hours together with cell, shows that CFU-GM is trapped in the surface of sample, and this point can be bright by the authentication of the red staining existing on sample surfaces.This accompanying drawing has also shown that approximately 75% cell is the VEGFR-2 positive, and form is circular.

Fig. 8 A is the sample of having hatched together with cell 7 days with 8B.As shown in Figure 8 A, the core of red staining shows that cell is present on sample, and these cells are the VEGFR-2 positive (Fig. 8 B, 100%), and by structurally endothelialization more of these cells shown in the spindle of cell.Fig. 9 A and 9B be comprise in its surface CD34 antibody coating the sample microphotograph of CMXD, this sample has been hatched 7 days together with cell, after hatching, makes this sample contact Tie-2 antibody.As shown in Figure 9 A, the core of red staining has shown has many cell adhesions in sample surfaces.As visible in the green fluorescence of cell transmitting, the cell that adheres to sample is also the Tie-2 positive (100%) (Fig. 9 B).Generally speaking, the VEGFR-2 existing in sample surfaces and adherent cell surface by many cell adhesions is with shown in Tie-2 receptor: cell was hatched after 7 days together with sample, and the sample that has applied CD34 antibody can be trapped in endotheliocyte their surface.In addition, 7 days time, on sample surfaces, exist 100% endotheliocyte to show that non-endotheliocyte may come off, or all adherent cell have all started to express endothelial cell marker in the time of the 7th day.

Figure 10 A-10C is the phase contrast microscope photo that is grown in endothelial cell growth culture medium the endothelial progenitor cells of 3 weeks.Figure 10 A showed cell has been broken up becomes ripe endotheliocyte, and this point can be proved by the intravascular space of the two-dimentional tubular structure at arrow place.Figure 10 B shows the three-dimensional cell stack that has multilamellar, and a cell is positioned on another cell, and this starts to form with regard to having confirmed the endotheliocyte of growth in time expand the report that is positioned at the layer on another layer.Figure 10 C is depicted as the CFU-GM of cultivating after kind of plate 3 weeks, and these cells have the outward appearance of endotheliocyte, and the CD34/FITC antibody green fluorescence existing as their surfaces proves, this accompanying drawing has confirmed that these cells are endotheliocyte.

Above-mentioned data show separates from the leukocyte of human blood and contains the positive CFU-GM of CD34, and these cells can be grown for mature endothelial cell and be easy to express surface endothelial cell antigens.(VEGFR-2 and Tie-2).These data also show on the surface that can adopt the medical treatment device these cell captures being applied in the present invention for the antibody of CFU-GM or stem cell surface antigen.

Embodiment 4

Apply fullerene and applied fullerene the body of stainless steel with anti-CD34 antibody and/or endothelial cell growth factor (ECGF) (Ang-2, VEGF)

Adopt following the method stainless steel stent and the disk that produce the functional Fullerene layer with binding antibody and/or somatomedin (, VEGF or Ang-2):

In the first step, with the surface of 0.5M HCl activation SST support or disk, described HCl has also removed lip-deep any passivation pollutant simultaneously.From activating bath, take out metal sample, with distilled water flushing, methanol is dried and dries at 75 DEG C.Then described support be impregnated in and contain oxidation fullerene (C ₆₀-O) toluene derivative solution in maximum 24 hours.Described oxidation fullerene can be incorporated into support by the Fe-O on support, Cr-O and Ni-O.Support is taken out from derivative bath, with toluene flushing, be then placed in Soxhlet Extractor 16 hours, remove the C of any physical absorption with fresh toluene ₆₀.Take out support, and dry and spend the night at 105 DEG C.This reaction has produced completely derivative support or the disk with fullerene monolayer.

In step 2, make decanedioic acid and thionyl chloride or sulfur oxychloride (SOCl ₂) reaction formation sebacoyl chloride, to form dialdehyde molecular solution.Gained sebacoyl chloride and the LiAI[t-O butyl of making as follows] ₃h and diethylene glycol dimethyl ether react to obtain 1,10-decanedioyl:

In step 3, on the support available from step 1 or disk, form N-methylpyrrole alkane derivatives.As follows, by following reaction, fullerene molecule is further derived: under nitrogen, in the toluene solution refluxing, by 1 in the fullerene of equimolar amounts and sarcosine and step 2,10-decanedioyl product reaction 48 hours, to obtain N-crassitude derivative fullerene stainless steel stent or disk.

Wash derivative stainless steel stent or disk to remove any chemical residue, for standard method binding antibody and/or (VEGF or Ang-2).Separate as described in Example 1 the CFU-GM in human blood, the fullerene disk that its contact is applied with anti-CD34 antibody.After hatching, remove growth medium, use PBS washing sample 2 times.With 2% paraformaldehyde (PFA) fixed cell 10 minutes, then, with PBS washing 3 times, wash 10 minutes, to guarantee to remove all fixatives at every turn.Each sample is at room temperature hatched 30 minutes together with lock solution, to seal all non-specific binding.By PBS washing sample 1 time, add the VEGFR-2 antibody incubation of dilution in 1: 100 to spend the night.Then use PBS washing sample 3 times, to guarantee to remove all primary antibodies.In each sample, add FITC coupling two anti-of doing dilution in 1: 100 with confining liquid, incubated at room 45 minutes on Belly Dancer device.After hatching, by PBS washing sample 3 times, wash once with the PBS that contains 0.1%Tween 20, then wash with PBS.With iodate the third ingot (PI) fixed sample, and observe under Laser Scanning Confocal Microscope.Figure 11 shows that the present invention combine CFU-GM coating the schematic diagram of rack surface of functional fullerene.Figure 12 A-12B be respectively with the coating of PI dyeing (12A) and anti-VEGFR-2/FITC-coupling antibody staining fullerene and without the microphotograph of the control sample of antibody.Figure 12 C and 12D are the microphotograpies that has applied the sample of fullerene/anti-CD34 antibody coating.As shown in the figure, the sample that is coated with anti-CD34 antibody contains the surperficial VEGFR-2 positive cells that adhere to more.

As described in Example 5, by contain or do not contain antibody coating fullerene sample implant yorker.Take out support and make histologic analysis, with 10% formalin buffer flushed zone support section 30 seconds, then fix until process by 10% formalin buffer.From each support, cut following 5 sections; Close on support 1mm place, support faces subterminal 1mm place, support middle section, rack far end 1mm place and support end 1mm place.By hematoxylin & Yihong (HE) and trichroism elastin laminin stained.Figure 13 A-13D is the support cross section microphotograph of implanting 4 weeks of taking out from coronary artery.The support that data show has applied fullerene (Figure 13 B and 13D) has suppressed the neointimal hyperplasia (blank support, Figure 13 A and 13C) at support position of living in compared with the control.

Embodiment 5

Pig air bag damage research: yorker childhood that the structural transplantation that has applied antibody is entered to heavy 25-30kg.Take care of animal according to " management of laboratory animal and guide for use " (Guide for the Care and Use of Laboratory Animals, NIH publication number 80-23, revises in 1985).After overnight fasting, make animal calmness (20mg/kg) with ketalar.Then use penthiobarbital (12mg/kg) induced anesthesia, animal is carried out to intubate, and connect the snorkel that gives oxygen and nitrogen mixture (1: 2[volume/volume]).Maintain anesthesia with the isoflurane of 0.5-2.5 volume %.Intramuscular injection 1,000mg procaine benzylpenicillin-G and benzathine benzylpenicillin-G (streptomycin) mixture is to provide the prevention of antibiosis disposition.

Under aseptic condition, carry out left neck artery otomy, 8F guide sheath is placed in to left neck artery.Give all animal 100IU heparin/kg body weight.In whole operation process, regularly append 2, the heparin medicine group (boluse) of 500IU, to remain on activated clotting time more than 300 seconds.6F guiding tube is imported by carotid sheath, be advanced to coronary ostium.In coronary artery, give, after 200 μ g nitroglycerine, to carry out angiography, with quantitative coronary angioradiographic system analysis image.3F Embolectomy catheters is inserted to near-end coronarius and be advanced into and select for implant frame and the exposed section of endothelium to far-end.The R support through applying that adds anti-CD34 antibody is inserted by guiding tube, and launch in the exposed section of coronary artery.With blank stainless steel stent or applied substrate but containing the support of antibody not in contrast.Taking support, tremulous pulse is implanted to left anterior descending coronary artery (LAD) or right coronary artery (RCA) or circumflex branch of coronary artery (Cx) by support as 1.1 ratio.Evaluate size and the position of support with angiography, take out guide sheath, closed two-layer skin.In process of the test, give the ASA of animal 300mg.

After implant frame, put to death animal 1,3,7,14 and 28 day time.First animal is calm and anesthesia as mentioned above.Take out the coronary artery of implant frame, at its near-end and far-end with the unsupported blood vessel of 1cm.Process the blood vessel of implant frame by three kinds of methods: histology, Immunohistochemical Method or scanning electron microscope method.

Detect for SABC, softly rinse the support 30 seconds of incision with 10% formalin, be placed in 10% formalin/PBS solution until processing.The support detecting for SABC with the PBS solution flushing appointment containing 2% paraformaldehyde (PFA) 30 seconds, then be placed in 2%PFA solution 15 minutes, washing and storing with PBS until use the SABC of rabbit human VEGFR-3 resistant-2 or mouse-anti-human T ie-2 antibody to detect.

Prepare by the following method the support for SEM: rinse 30 seconds by 10% formalin buffer, then in the 0.1M sodium cacodylate buffer liquid that contains 2.5% glutaraldehyde and 2%PFA, fixedly spend the night.Then use dimethyl arsenic acid buffer liquid washing sample three times, and its cleaning is spent the night.Complete fixing post processing by following steps: with the 0.1M dimethyl arsenic acid buffer liquid processing of 1% Osmic acid. (Sigma), then use ethanol dehydration (30% ethanol, 50%, 70%, 85%, 95%, 100%, 100%), use subsequently CO ₂carry out critical point drying.After dry, sample carried out to metal spraying, and observe under SEM.(" coronary artery of normal pig is adopted to the Palmatz-Schatz support applying through heparin to reduce or remit thrombotic support mould bases ", Reduction in thrombotic events with heparin-coated Palmaz-Schatz stent in normal porcine coronary arteries, Circulation 93:423-430, includes in herein as a reference).

Rinse for the support section of histologic analysis 30 seconds by 10% formalin buffer, then fix until process by 10% formalin buffer.Cut following 5 sections from each support; Close on support 1mm place, support faces subterminal 1mm place, support middle section, rack far end 1mm place and support end 1mm place.By hematoxylin & Yihong (HE) and trichroism elastin laminin stained.

The taking-up support that Figure 14 A-14G observes under scanning electron microscope for implanting latter 1 hour (Figure 14 A and 14B) and 48 hours (Figure 14 C-14G).Microphotograph has clearly shown the support (14B with glucosan/anti-CD34 antibody applies, 14E-G) catch endothelial progenitor cells, this point can by with contrasting by contrast of applying with glucosan, section in 48 hours is being spindle outward appearance (14A, 14C and 14D) compared with the lower cell of high power (400X).

The transverse section of porcine coronary separator also shows compared with the control (blank rustless steel 14H and 14I; The 14J and the 14K that apply with glucosan), apply (14L, 14M) with glucosan-anti-CD34 antibody and make neointimal hyperplasia (thickness of arterial smooth muscle layer) be subject to significant inhibition.With fullerene apply stent graft suppressed better neointimal hyperplasia (as shown in Figure 13 B-13D) compared with blank stainless steel stent.

Figure 15 A and 15B are respectively after 48 hours long the applying but copolymerization Jiao microphotograph of the anti-CD34 antibody support that surface applies without the support separator of antibody with glucosan-blood plasma with glucosan-blood plasma of the 18mm that takes out.Described support was once implanted the coronary artery of male York children pig.The support taking out is carried out to SABC is processed and VEGFR-2 dyes, then with two anti-processing of FITC coupling with study under Laser Scanning Confocal Microscope.Compared with Figure 15 B and 15C have shown and lack endothelium completely with the support containing antibody not, the support that contains antibody is covered by endotheliocyte, and this green fluorescence by this section is confirmed (Figure 15 A).

Embodiment 6

Endothelial cell growth factor (ECGF) is mixed to the substrate of antibody that put on fixing of support: the step that the antibody of anti-endothelial progenitor cells surface antigen is fixed on to the biocompatible matrix that puts on endovascular stent has been described below, endothelial cell growth factor (ECGF) be adsorbed in subsequently support taking promote circulation endothelium progenitor cell adhere to and in the time contact with blood maturation as functional endothelium.

Apposition: adopted method known to those skilled in the art, with plasma deposition technique process stainless steel stent with rack surface import amino functional.Employing is called the standard method of water-soluble carbodiimide coupling chemical method, under aqueous conditions, carboxyl group functional glucosan layer is incorporated into the amino-functional layer being deposited on support by glucosan (CMDX) carboxyl of activation, the amino being positioned under described aqueous conditions on plasma deposition layer forms amido link between plasmasphere and functionality CDMX.

The immobilization of antibody: by hatching in the acidic buffer solution of moisture water-soluble carbodiimide chemical reagent, by the antibody of anti-endothelial progenitor cells surface antigen (for example Mus monoclonal anti-human CD34) covalent coupling in CDMX apply support.

The absorption of somatomedin: monoclonal anti-human CD34 is fixed on after the CMDX substrate that puts on support, by described device be incubated in contain suitable concentration endothelial cell growth factor (ECGF) (for example, angiopoietin-2) aqueous solution in, make somatomedin be absorbed into CMDX substrate.Rinse treated device with normal saline solution, and be stored in Hydrazoic acid,sodium salt preservative agent.

Employing standard Angiography, in the time said apparatus being implanted to porcine coronary and contacted human blood, produce and strengthened circulation endothelium progenitor cell and catch and be attached to treated or drug delivery medical device surface and promote that cell maturation is the effect of functional endothelial tissue.The Rapid Establishment of functional endothelial tissue can reduce the device thrombosis causing and the degree that regulates neointimal hyperplasia.

Embodiment 7

Endothelial cell growth factor (ECGF) and antibody are fixed on support: described below the antibody of anti-endothelial progenitor cells cell surface antigen and somatomedin be fixed on the biocompatible matrix that puts on endovascular stent, thereby in the time contacting with blood, promote circulation endothelium progenitor cell adhere to and ripe be functional endothelial tissue step.

Apposition: apposition has used method known to those skilled in the art, with plasma deposition technique process stainless steel stent to import amino functional on rack surface.Employing is called the standard method of water-soluble carbodiimide coupling chemical method, under aqueous conditions, carboxyl group functional glucosan layer is incorporated into the amino-functional layer being deposited on support by activated dextran (CMDX) carboxyl, the amino being positioned under described aqueous conditions on plasma deposition layer forms amido link between plasmasphere and functionality CDMX.

The immobilization of antibody and somatomedin: by under acid condition, by etc. the antibody (for example Mus monoclonal anti-human CD34) of anti-endothelial progenitor cells surface antigen of molar concentration and endothelial cell growth factor (ECGF) (for example, angiopoietin-2) hatch in water-soluble carbodiimide reagent solution, make itself and the support covalent coupling applying with CDMX.Rinse treated device with normal saline solution, and be stored in Hydrazoic acid,sodium salt preservative agent.

Employing standard Angiography, in the time said apparatus being implanted to porcine coronary and contacted human blood, produce and strengthened circulation endothelium progenitor cell and catch and be attached to treated or drug delivery medical device surface and promote that cell maturation is the effect of functional endothelial tissue.The Rapid Establishment of functional endothelial tissue can reduce the device thrombosis causing and the degree that regulates neointimal hyperplasia.

Embodiment 8

The micromolecule functionalization of support: separate and obtain endothelial progenitor cells as described in Example 1.Cell is inoculated in to the slide of fibronectin covering and it is grown 7 days in EBM-2 culture medium.Fixed cell is also used the endothelial cell specific agglutinin dyeing (Ulex Europaeus Uea1) of iodate the third ingot (PI) and FITC coupling.The results are shown in Figure 16 A and 16B of these tests.These accompanying drawings have shown that endothelial progenitor cells is incorporated into the slide that fibronectin applies, and these cells are expressed the part that has this agglutinin on their surface.

Embodiment 9

With the bicistronic mRNA carrier By Transfecting Porcine endothelial progenitor cells (EPC) of coding vasodilation compound and unique cell surface marker (MHC-I of truncate).MHC-I can be fixed in the identification of the specific antibody on dummy in blood vessel.The support that antibody is applied is implanted in porcine coronary, then the EPC of hereditary change is implanted to pig.Due to antibody-AI, EPC is trapped on the support through applying, and in stent strut, forms endothelial cell monolayer.Captive cell can be secreted vasodilation, the increase far-end blood flow of expressing and trigger positivity and mould.

the selection of plasmid: Miltenyi Biotec (Germany) has developed and has contained pMASCSK ^kthe MACSelect K System of plasmid vector.Described pMACSK.II plasmid is the bicistronic mRNA carrier (5229bp) that one contains multiple clone site (MCS), and clone has the cDNA of coding prostacyclin synthase and the gene of coding Mus MHC I type molecule H-2K therein.Developing this system is in order to select the cell for transfection, using the MHC molecule of truncate as selected marker.The expression of natural H-2K is limited to (for example, AKRiJA or CBNJ) in some rare Mus strain, therefore for H-2K ^kshould substantially can not there is not irrelevant reaction with other surface antigen in the monoclonal antibody (Miltenyi Biotec) of surface protein.

Assessment with the cross reactivity of whole blood: in order to ensure anti-H-2K ^kantibody not with pig whole blood in cellular component generation cross reaction, make the anti-H-2K antibody response of whole blood and FITC coupling, and carry out whole blood facs analysis (Beckman Coulter Cytomics FC 500).With expressing H-2K ^kthe whole blood of Mus spleen fibroblast strain AKRIJASp (American type culture collection, American Type Culture Collection, ATCC) " strengthening " (" spiked ") of surface antigen is as positive control.

Fibroblastic cultivation: AKR/JA.Sp fibroblast is incubated in the T-75 plastic bottle of uncoated (Sarstedt, Montreal), condition of culture is: 37 DEG C, 5%CO ₂, culture medium is Da Erbaike (family name) improvement Iger (family name) culture medium (DMEM) that contains 4mM L-glutamine, 4500mg/L glucose, 1mM Sodium Pyruvate, 1500mg/L sodium bicarbonate and 10% hyclone.Carry out cell separation with trypsin/EDTA (Invitrogen).By immunohistochemical analysis, use fluorescently-labeled H-2K ^kantibody has confirmed H-2K ^kexpression.In brief, with 0.5 × 10 ⁶individual cell/cm ²cell is inoculated in the band chamber slide (chamber slide) of 2 hole uncoated.In the time of the 1st, 2,3 and 4 days, with 2% paraformaldehyde fixed culture, and with H-2K antibody (the Miltenyi Biotec of FITC coupling, Germany) and core labelling iodate the third ingot (PI) (Vectashield Mounting Medium, Vector Laboratories) dyeing.Adopt Laser Scanning Confocal Microscope (Nikon Eclipse E800-Biorad Radiance 2 100) to analyze with quantitative.Human fibroblasts is used as to negative control.

The not analysis of adherent cell: H-2K ^kthe reservation of surface protein is the feature of the AKRIJA.Sp cell of non-cohesive form, adopts the feasibility of this system when confirming that blood exists.As mentioned above by cell culture in the T-75 of uncoated bottle.Made the cell detachment adhering to trypsin/EDTA at the 4th day, and with the H-2K of FITC coupling ^kantibody and facs analysis (Beckman Coulter Cytomics FC500) are measured and are expressed H-2K ^kthe cell quantity of surface protein.Using the Mus IGg2 α isotype of FITC labelling as negative control.

The structure of plasmid: utilize BamHl and the restricted sequence of HindIII in multiple clone site that the cDNA of coding prostacyclin synthase is cloned into bicistronic mRNA carrier pMACS K ^k.II (Miltenyi Biotec, Germany).Use the cDNA of 1153 base pairs that contain prostacyclin synthase gene and pVAX-1 that are arranged in plasmid construction thing.Under existing, selective agent ampicillin (50ng/ml) carry out the colibacillary conversion of HG70.

Be used for the full-length cDNA of people α-CGRP available from open biosystem (Open Biosystems, the classification number #MHS 1768-9 1,441 17 of plasmid vector pPCR-Script Amp SK (+); Huntsville AL).Then this fragment and BamHl/EcoRl are connected into bicistronic plasmid carrier pMACS K.II.Transform JM109 escherichia coli to obtain a large amount of described plasmids.

The transfection of EPC: with Ficoll gradient centrifugation enrichment pig mononuclear cell from pig whole blood, and with above-mentioned enrichment medium separation EPC.Cultivate after 7 days, by the method (Amaxa Nucleofector, Germany) of core perforation, with the bicistronic plasmid carrier transfection EPC that contains metastatic gene, described metastatic gene comprises α-CGRP or prostacyclin synthase.Utilize reporter gene and endothelial nitric oxide synthase (eNOS) in pVAXt plasmid, obtained the EPC Efficiency of Electroporation (data are not shown) of > 70%.To successful transfection and express H-2K ^kthe EPC of surface protein carries out purification, and removes test kit (MACS Dead cell removal kit), MACSelect K with MACS dead cell ^kmicroballon (MACSelect K ^kmicroBead) separate with MS detached dowel (MS Separation Column) (Miltenyi Biotec).MACSelect K ^kmicroballon is biodegradable, and it disappeared with cell culture in 24 hours.

The mensuration that vasodilation is expressed:

The mensuration of prostacyclin synthase activity: after 2 days, keep cultivating the EPC through transfection in transfection.Change culture medium, and assess the activity of prostacyclin synthase according to the level of prostacyclin synthase metabolite 6-ketone-PGF1α (6-keto-PGFIcu) in manufacturer's radio immunoassay for explanation (Amersham Corp.) mensuration culture medium.

The mensuration of α-CGRP activity: measure the expression of α-CGRP in transfectional cell with Immunohistochemical Method color reagent box (Bachem USA).At-10 DEG C, fix with methanol the EPC5 minute through transfection cultivating 3 days.Wash described cell air-dry.Fixing cell is hatched in the PBS of 0.5% hydrogen peroxide solution to 7 minutes with deactivation endogenous peroxide activity.Cell is hatched in serum confining liquid to 20 minutes with sealing non-specific binding.Then use anti-α-CGRP primary antibodie (rabbit monoclonal, Bachem) with three kinds of dilution factors: within 1: 100,1: 200 and 1: 500, to process cell 2 hours.Then wash slide, make it contact biotinylated two and resist 30 minutes.Then rinse cell and use HRP-streptavidin complex to process 30 minutes.With after PBS washing, make cells contacting substrate-chromogen mixture 3 minutes.Add deionized water with cessation reaction.With MayerShi haematoxylin redyeing slide 3 minutes.Then rinse slide with tap water, be placed in PBS until become blue, use distilled water rinsing.With 95% and 100% ethanol and dimethylbenzene, slide is dewatered again.Covered checking under light microscopic on slide.

The support applying with antibody: foregoing with glucosan and anti-H-2K ^kantibody applies stainless steel stent (9mm is long).

Cells in vivo is caught: all tests are all carried out in (> 30kg) in the children pig of male York.Obtain arterial passageway by the arteriotomy of carrying out on left neck artery.In coronary artery, give after 200pg nitroglycerine, obtain coronary angiography, and carry out online quantitative coronary angiography and measure.With the supports of 1.1: 1 to blood vessel than support is launched in the proximal section of LAD convolution or right coronary artery at random.After implanting, just in intravascular, give 200pg nitroglycerine.Then carry out intravascular ultrasound (IVUS) to measure external caliber, prop up using the distally of stent with remote edge as far-end and near-end reference.Utilize prototype series connection balloon catheter (Cordis Corporation) to complete through the giving of transfectional cell, the bicistronic mRNA carrier transfection of coding prostacyclin synthase or α-CGRP for described cell.This conduit by be positioned at two of apparatus adjacent far-end highly the air bags of laminating form, by same air-filled pore this two air bags that expand.Once after expanding, separate the long blood vessel section of 1.0cm between two air bags, to produce a regional perfusion chamber.Provide far-end blood flow by central chamber, and the chamber that is fed into solution or passes through two separation is by whole solution suction chamber.Filling cavity ends near of far-end air bag, and discharge side ends at a near opening of of proximal balloon.Series connection balloon catheter is reached forward to the position of implant frame, and airbag inflation is arrived to 25psi (1.7 atmospheric pressure).Send saline by fill orifice, until do not contain blood in the section separating.The tremulous pulse section of implant frame is carried out to random packet to accept perfusion of saline or cell is sent.In 10 minutes, be total up to the 2ml cell suspension of 3 × 10 EPC with the irrigation rate of 200pL/ minute, then hatch 10 minutes.Then closed position of implementing arteriotomy, restores animal.Letting animals feed 28 days after cell is processed.Coprocessing 34 animals (10 is saline control, and 14 is prostacyclin synthase, and 14 is α-CGRP).Delivery of cells after 1 hour is killed two animals of every group.Separate the section of implant frame, and prepare the tremulous pulse for the implant frame through rinsing of SEM by following steps: fix 30 seconds at 10% formalin buffer PBS, then further in the 0.1M sodium cacodylate buffer liquid (Sigma) that contains 2.5% glutaraldehyde (BDH Inc.) and 2%PFA, fixedly spend the night.Complete fixing post processing by following steps: with the 0.1M arsenate buffer processing of 1% Osmic acid. (Sigma), then use Gradient elution using ethanol, use subsequently CO ₂carry out critical point drying.After dry, sample is carried out metal spraying and whether has in the lower observation of scanning electron microscope (SEM) cell that is incorporated into stent strut.After implant frame the 5th day put to death two animals of prostacyclin synthase group and two animals of α-CGRP group.The tremulous pulse section containing implant frame taking out is placed in to 10% formalin/PBS solution, until carry out normal structure chemical analysis.Cut 5 sections from each support; Close on support 1mm place, support faces subterminal 1mm place, support middle section, rack far end 1mm place and support end 1mm place.Section is dyeed with hematoxylin & Yihong (HE) and trichroism elastin laminin.Measure inflammation [section's Milunovich index, Kornowski Score (0-3)] index to assess the rejection sign to input cell.Carrying out (approximately 28 days) after exponential process, anesthetized animal also carries out coronary angiography by right coronary artery angiotomy.Carry out quantitative coronary angiography, utilize IVUS to carry out blood vessel inquiry, record the variation of external caliber with standard clinical algorithm.

Embodiment 10

Mammalian cells in vitro transfection for vascular remodeling: adopt the method for electroporation that bicistronic plasmid is transfected into endothelial progenitor cells, described plasmid comprises coding and is responsible for the protein of adenosine generation and the gene of prostate specific epicyte protein.Two kinds of genes are subject to them to initiate self sub control separately, thereby make gene be able to constructive expression.

Build following carrier with being similar to above-mentioned method, the gene that described carrier comprises coding prostate specific membrane albumen and its natural promoter, and the gene of coding VEGF, these two kinds of genes are arranged in series in same expression vector.Can be by this plasmid construction thing for the cell for patient described in transfection embodiment 9, mammalian cell.

Claims (16)

1. treat a therapy system for patient disease, described system comprises the following described patient's who needs respectively this treatment component:

(i) mammalian cell of hereditary change, described cell comprises coding through genetically engineered cell membrane labelled molecule and the exogenous nucleic acid of at least one therapeutic gene product; Mammalian cell energy presentation markup molecule and the therapeutic gene product of described hereditary change, and described cell gives described patient so that it is present in described patient's circulating, and wherein said cell membrane labelled molecule is selected from: BMP, prostatic cell memebrane protein or H-2Kk surface protein; With

(ii) for implanting described patient's medical treatment device, described device comprises coating; Described coating comprises load the biocompatible matrix of at least one part, described substrate comprises: synthetic material, comprises polyurethane, block polyurethane-carbamide/heparin, Poly-L-lactide, cellulose esters, Polyethylene Glycol, polyvinyl acetate, glucosan or gelatin; And/or naturally occurring material, wherein said at least one ligand specificity identification in conjunction with described cell membrane labelled molecule, and described part is selected from the combination of antibody, antibody fragment, antibody and antibody fragment,

Wherein, in the use of described therapy system, the mammalian cell original position of the described hereditary change in the circulation giving is separately incorporated at least one therapeutic gene product described in described medical treatment device expression and secretion, and wherein said therapeutic gene product is selected from prostacyclin, calcitonin-gene-related peptide, VEGF, angiogenesis factor, anti-angiogenesis or fibroblast growth factor.

2. therapy system as claimed in claim 1, is characterized in that, described medical treatment device comprises any device of the implantable organ that comprises tract or body part.

3. therapy system as claimed in claim 1, it is characterized in that, described medical treatment device is selected from: support, stent graft, synthetic blood vessel graft, cardiac valve, conduit, vascular repair screen, pacemaker, pacemaker lead, defibrillator, in hole, ovum garden patent (PFO) every closing device, vascular clamp, vascular aneurysms dead lock, hemodialysis graft, hemodialysis catheter, chamber diverter, aortic blood tuberculation graft device or assembly, venous valve, suture, blood vessel anastomosis clamp, remain-type vein or ductus arteriosus, vagina vasorum and drug conveying mouth.

4. therapy system as claimed in claim 3, is characterized in that, described medical treatment device is the support that comprises following material: rustless steel, Nitinol (NiTi) or evanohm and Biodegradable material; Synthetic blood vessel graft can be made by crosslinked PVA hydrogel, politef (PTFE), expanded polytetrafluoroethyltoe (ePTFE), porous type high density polyethylene (HDPE) (HDPE), polyurethane and poly terephthalic acid vinyl acetate or Biodegradable material.

5. therapy system as claimed in claim 1, is characterized in that, the mammalian cell original position of described hereditary change forms fused cell layer.

6. therapy system as claimed in claim 1, medicine or compound that it also comprises the mammalian cell expression for stimulating described hereditary change and/or secretes described therapeutic gene product.

7. therapy system as claimed in claim 1, is characterized in that, the mammalian cell of described hereditary change is autologous, allogeneic or heterogenous cell.

8. therapy system as claimed in claim 7, is characterized in that, described autologous, allogeneic or heterogenous cell are endothelial progenitor cells or mature endothelial cell, fibroblast or sarcoplast.

9. therapy system as claimed in claim 1, is characterized in that, the described exogenous nucleic acid being present in the mammalian cell of described hereditary change comprises DNA or RNA molecule, at least one gene that described molecule comprises at least one therapeutic gene product of encoding.

10. therapy system as claimed in claim 1, is characterized in that, described exogenous nucleic acid is exchromosomal DNA.

11. therapy system as claimed in claim 10, is characterized in that, described DNA molecular comprises plasmid.

12. therapy system as claimed in claim 10, is characterized in that, described exchromosomal DNA comprises that regulation and control box, cell membrane specific gene and at least one coding are used for the treatment of the gene of the peptide of disease.

13. therapy system as claimed in claim 1, is characterized in that, described part is combination, peptide or the micromolecule of antibody, antibody fragment, antibody and antibody fragment.

14. therapy system as claimed in claim 1, is characterized in that, described medical treatment device comprises dummy, and described dummy comprises: (a) have outer surface and the carrier film that contacts blood surface; (b) be coated on the lip-deep ground floor cross-linked polymer compound of contact blood of described carrier film; (c) be coated on the second layer on described ground floor, the described second layer contains in vivo can be in conjunction with at least one part of the mammalian cell of the described hereditary change of circulation.

15. therapy system as claimed in claim 1, is characterized in that, described naturally occurring material is basement membrane composition.

16. therapy system as claimed in claim 15, is characterized in that, described basement membrane composition is selected from: collagen, elastin laminin, tropoelastin, laminin, fibronectin, vitronectin, heparin, fibrin, cellulose and amorphous carbon.

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