Implantable materials in contact with blood and uses thereof

Abstract

The present invention is directed to blood compatible materials, implantable devices comprising said materials, methods for the preparation of such materials, and medical devices coated therewith, as well as uses thereof for anti-thrombotic and / or cell proliferation aspects.

Description

[Technical Field]

[0001] The present invention relates to implantable materials that come into contact with blood, and their preparation and application, for example in implantable devices such as vascular grafts, shunts, patches, stents, valves, etc. In particular, the present invention relates to biocompatible materials and implantable artificial devices (endoprostheses) useful for the surgical or therapeutic treatment of humans or animals (especially of cardiovascular disease, especially cardiovascular ischemic, degenerative or congenital disease, e.g., coronary artery disease or peripheral vascular disease, or access surgery for dialysis patients, pediatric cardiovascular malformations, trauma, and reparative surgery). [Background technology]

[0002] Cardiovascular ischemic disease is the leading cause of death in the Western world, and vascular grafts with small internal diameters (IDs) remain a challenge for material scientists. The primary failure mechanisms associated with commercially available grafts include early thrombosis / occlusion and lack of rapid and confluent endothelialization, as well as late intimal hyperplasia that can lead to stenosis and occlusion.

[0003] This suggests the need for the development of improved artificial vascular conduits that do not cause pathological reactions after implantation. The graft should resemble native blood vessels in terms of biocompatibility, antithrombogenicity, shape, size, and mechanical properties, compliance, regeneration and growth potential, and resistance to infection. The use of naturally derived materials (e.g., collagen or decellularized tissue matrix) carries the risk of immunological reactions leading to premature degradation, which can cause aneurysms and rupture. The fabrication of fully cell-derived grafts is very time-consuming, requiring approximately six months. Therefore, in clinical practice, surgeries using small-diameter vascular grafts (less than 6 mm) are primarily performed with autologous arteries or veins, which may be pathological or may have been used in previous surgeries. Therefore, there is a strong demand for readily available small-diameter vascular grafts. Synthetic polymers offer the advantage of being manufacturable on a large scale with controllable physical and chemical properties, such as strength, degradation rate, and reproducible microstructure. Commercially available grafts are made of non-degradable polymers such as polyethylene poly(ethylene terephthalate) (PET), Dacron®, or expanded poly(tetrafluoroethylene) (ePTFE) or poly(urethane) (PU), or others, and are widely used as non-degradable synthetic vascular grafts.

[0004] While it has been successfully used to replace large-diameter blood vessels, it has failed as a small-diameter prosthesis (ID < 6 mm) required for smaller vessels and / or microvascular anastomoses. Vascular grafts with small internal diameters present a challenge for material scientists. They have the potential to be employed in the treatment of cardiovascular ischemic diseases such as coronary artery disease or peripheral vascular disease, which remain the leading cause of non-communicable death in the Western world. Additionally, they may be required for the treatment of end-stage renal disease in access surgery. When possible, bypass surgery is performed with autologous arteries or veins because synthetic vascular grafts have shown poor clinical outcomes when used with small-diameter grafts (ID < 6 mm). In contrast to large-diameter grafts, prostheses with internal diameters less than 6 mm are prone to hemodynamic flow disturbances and increased thrombotic complications. Therefore, endothelialization of small-diameter grafts must form an interface compatible with hemodynamic and functional processes. The primary failure mechanisms are premature thrombosis (cell-surface interactions) and the lack of rapid, confluent endothelialization. This failure is primarily the result of an unfavorable healing process involving surface thrombogenicity due to the lack of endothelial cells and anastomotic intimal hyperplasia caused by hemodynamic disturbances. Intimal hyperplasia can subsequently lead to stenosis and occlusion, often necessitating reoperation or, in distal cases, amputation. All of these limitations also apply to any device (e.g., valve, VAD) that is implanted in contact with blood. As a result of the above, synthetic grafts are not used in coronary artery surgery. Therefore, the development of an ideal small-diameter prosthesis remains a major challenge in vascular research.

[0005] Numerous surface coatings have been developed over the past 30 years to improve the hemocompatibility of biomaterials, and as reviewed in Biran et al., 2016, Adv. Drug Deliv. Rev., 112, pp. 12-23, the anticoagulant heparin has been employed as a covalently immobilized surface coating on various medical devices to improve hemocompatibility.

[0006] To further improve the hemocompatibility (i.e., heparin conjugation) of expanded poly(tetrafluoroethylene) (ePTFE), a so-called non-treated surface, and / or polycarbonate-urethane (PCU) surfaces, numerous surface modification strategies (e.g., chemical immobilization, physical adsorption, and plasma treatment) have been investigated. It has been shown that PCU surface functionalization achieved using plasma treatment is significantly more effective at generating functional amine groups on the surface than alternative methods, such as aminolysis and physical adsorption via polydopamine coating. These functional amine groups, then grafted onto the surface of a vascular graft, can be used for subsequent heparin conjugation via reductive amination, enabling terminal immobilization of heparin molecules on the graft surface (Qiu et al., 2017, Acta Biomater., 51, pp. 138–147). Conjugation of heparin by plasma treatment followed by reductive amination has been demonstrated to advantageously provide not only higher surface density but also better stability and antithrombogenic activity, and improve the performance of vascular grafts with respect to patency and the early stages of endothelialization and graft integration.

[0007] Biodegradable polymers have also been investigated for their potential as scaffolds for fabricating small-diameter arterial bypass grafts. Two methods for aminolysis of electrospun poly-L-lactide-co-caprolactone (PLCL) microfiber grafts were compared: plasma treatment and fmoc-PEG-diamine insertion, which allowed for terminal heparin conjugation to PLCL scaffolds, optimizing heparin density and bioactivity in biodegradable electrospun PLCL microfiber grafts (Hsieh et al., 2017, Biomed. Mater., 12, 6). This study concluded that plasma treatment was superior in terms of initial heparin density, while chemical introduction of amino groups for terminal heparin conjugation (via fmoc-PEG-diamine insertion) caused partial fiber erosion, dissolution, and structural collapse, while inducing minimal polymer chain degradation. However, plasma activation of surfaces is very aggressive towards the support material and often causes undesirable defects in the structure and mechanical properties of the implantable material, and may even prevent further biomedical uses of biodegradable materials such as PCL (polycaprolactone).

[0008] Gao et al., 2018, Regen. Biomater., 5, pp. 105-114, recently prepared small-diameter vascular grafts based on a matrix of electrospun poly(ε-caprolactone) that was subsequently coated with alternating layers of selenium-containing catalyst-organoselenium-modified polyethyleneimine and heparin (layer-by-layer (LbL) assembly) to combine the effects of heparinization and catalytic NO production to stimulate tissue regeneration, angiogenesis, and vascular remodeling processes. In this assembly, heparin was used as the polyanion for LbL formation. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 4,613,665 [Non-patent literature]

[0010] [Non-licensed Document 1] Biran, 2016, Adv. Drug Deliv. Rev., 112, pp. 12-23 [Non-licensed Document 2] Qiu, 2017, Acta Biomater, 51, pp. 138-147 [Non-licensed Document 3] Hsieh, 2017, Biomed. Mater., 12, 6 pages [Non-licensed Document 4] Gao, 2018, Regen. Biomater., 5, pp. 105-114 [Non-licensed Document 5] Virk, 2019, Curr. Cardiol. Rep., 21, 36 pages [Non-licensed Document 6] L'Heureux, 2006, Nat. Med., 12, pp. 361-365 [Non-licensed Document 7] Channasanon, 2007, J. Colloid Interface Sci., 316, pp. 331-343. [Non-licensed Document 8] Cui, 2010, Adv. Funct. Mater., 20, pp. 3303-3312. [Non-licensed Document 9] Graisuwan, 2012, J. Colloid Interface Sci., pages 376, 177~188 [Non-licensed Document 10] Martins, 2014, Int. J. Mol. Sci., 15, pp. 20800-20832 [Non-licensed Document 11] Pfeiffer, 2014, J. Biomed. Mater. Res., 102A, pp. 4500-4509. [Non-licensed Document 12] Miranda, 2020, Antibiotics, 9, 174 pages [Non-licensed Document 13] Nottelet, 2009, J. Biomed. Mater. Res., 89A, pp. 865-875. [Non-licensed Document 14] de Valence, 2012, Acta Biomater, 8, pp. 3914-3920. [Non-licensed Document 15] Lian, 2003, J. Vasc. Surg., 37, pp. 472-80 [Non-licensed Document 16] Picart, 2015, Layer-by-Layer Films for Biomedical Applications, C. Picart, F. Caruso and J.-C. Voegel (eds.), Wiley-VCH: Weinheim, Germany [Non-licensed Document 17] El-Khouri, 2011, Functional Polymeric Ultrathin Films, R. Advincula and W. Knoll (eds.) Wiley-VCH: Weinheim, Germany [Non-licensed Document 18] Mourya, 2009, J. Mater. Sci. Mater. Med., 20, pp. 1057-79. [Non-licensed Document 19] Malik, 2018, Int. J. Nanomedicine, 13, pp. 7959-7970. [Non-licensed Document 20] Kulkarni, 2017, Carbohydr. Polym., 157, pp. 875-902. [Non-licensed Document 21] Biran, 2016, Adv. Drug Deliv. Rev., 112, pp. 12-23 [Non-licensed Document 22] Verheul, 2008 Biomaterials, 29, pp. 3642-3649. [Non-licensed Document 23] Martins, 2013, Carbohydr. Res., 381, pp. 153-160. Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, there is a need to discover suitable materials and / or coatings that allow for the preparation of small diameter vascular grafts that provide early, transient antithrombotic properties that prevent early thrombotic events and allow early, rapid, and confluent endothelialization of the graft. [Means for solving the problem]

[0012] The present invention relates to the unexpected discovery of novel biocompatible materials suitable for in vivo tissue regeneration, enabling the preparation of small-diameter grafts (1-6 mm inner diameter) useful in coronary artery bypass grafts (CABG), as described, for example, in Virk et al., 2019, Curr. Cardiol. Rep., 21, 36. These novel materials and coatings exhibit mechanical properties suitable for matching arterial compliance with strain and stress better than native arteries, enhancing suture retention and preventing aneurysm formation and rupture, thereby enabling their use in vascular grafts, shunts, patches, endoprostheses, valves, and any implantable device that comes into contact with blood. The method developed to prepare such materials offers the key advantages of producing materials with highly reproducible structural properties compared to chemical or plasma functionalization, and is more time-efficient than graft preparation methods requiring in vitro cellular manipulation, such as extracellular matrix formation based on cell type, ingrowth, and duration. (L'Heureux et al., 2006, Nat. Med., 12, pp. 361-365).

[0013] According to another aspect, the method of the present invention confers extended anticoagulant properties to the material. Specifically, the material of the present invention, when present on the luminal surface of a vascular patch or graft, provides an unexpected, long-lasting antithrombotic effect, which prevents initial thrombus formation or occlusion. According to certain embodiments, the material of the present invention, when biodegradable, induces a host response involving cell growth and de novo ECM (extracellular matrix) formation in vivo.

[0014] Furthermore, according to another specific embodiment, when a multilayer polymer layer-by-layer (LbL) coating of the material of the present invention comprises at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as the outer layer pair of the coating, the material of the present invention further exhibits the advantage of imparting advantageous cell attachment and growth properties by inducing rapid endothelialization due to a high affinity for endothelial cell growth on the surface. Thus, the material and method of the present invention make it possible to achieve the surprising property of prolonged anticoagulant properties while maintaining cell-attracting properties.

[0015] Another particularly advantageous and non-obvious aspect is the discovery of compositions and process steps that allow for the combination of the above functions on heterogeneous substrate surfaces without the use of aggressive coupling reactions that would chemically modify the surface or alter the mechanical properties of the surface.

[0016] Thus, the materials and methods of the present invention are expected to have beneficial effects with respect to any biocompatible implantable material intended to come into contact with blood upon implantation, particularly materials for use in implantable devices.

[0017] One aspect of the present invention provides a blood-compatible material comprising: (i) a substrate comprising a biocompatible nonwoven fiber; and (ii) a heparinized multilayer polymer layer-by-layer (LbL) coating on the biocompatible nonwoven fiber, the multilayer polymer coating comprising at least one layer pair of an anionic polymer layer and a cationic polymer layer, the multilayer polymer layer-by-layer (LbL) coating being functionalized with heparin on an outer layer pair of the multilayer polymer coating by terminal functionalization, wherein the anionic polymer is selected from heparin, poly(sodium 4-styrenesulfonate) (PSS), or a mixture thereof, and the cationic polymer is selected from chitosan or an analog thereof, and poly(allylamine hydrochloride) (PAH), or a mixture thereof.

[0018] Another aspect of the invention relates to implantable devices comprising the material according to the invention.

[0019] Another aspect of the invention relates to extracorporeal circulation devices such as membranes and catheters, oxygenators, and hemodialysis / filtration systems comprising the material of the invention.

[0020] Another aspect of the present invention relates to a method for preparing the blood compatible material according to the present invention.

[0021] Another aspect of the present invention relates to methods for preparing the implantable devices of the present invention.

[0022] Another aspect of the invention relates to a method of treating cardiovascular ischemic disease, e.g., coronary artery disease or peripheral vascular disease, pediatric cardiovascular malformation, trauma, or organ defect, e.g., end-stage renal disease (ESRD) or failure, by reparative surgery in a subject in need thereof, comprising implantation of an implantable device comprising a material of the invention in contact with the blood of the subject.

[0023] Another aspect of the invention relates to the use of the material according to the invention or the method according to the invention for the preparation of an implantable device or an extracorporeal circulation device that comes into contact with blood. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows the chemical structures of (a) poly(allylamine hydrochloride) (PAH), (b) poly(sodium 4-styrenesulfonate) (PSS), (c) fully deacylated chitosan (CHI), (d) dextran sulfate sodium salt (DS), (e) poly(L-lysine) (PLL), and (f) N,N,N-trimethylchitosan (TMC). [Figure 2A] 1 is a schematic diagram illustrating the structure of a blood-compatible material of the present invention, comprising (i) a substrate (1) comprising biocompatible nonwoven fibers; (ii) a heparinized multilayer polymeric layer-by-layer (LbL) coating of the present invention (2); and an optional pre-coating deposit (3), in which heparin (4) is end-functionalized to the multilayer polymeric coating (2). [Figure 2B] 1 is a schematic diagram illustrating the structure of a blood-compatible material of the present invention, comprising: i) a substrate (1) comprising biocompatible nonwoven fibers; (ii) a heparinized multilayer polymer layer-by-layer (LbL) coating (2) of the present invention; and an optional pre-coating deposit (3), wherein the multilayer polymer coating (2) comprises at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as an outer layer pair (21) forming a heparinized substrate for terminal functionalization of heparin (4), and an underlying layer of the multilayer polymer coating formed by another polymer layer pair (22) of the present invention. [Figure 2C]1 is a schematic diagram illustrating the structure of a blood-compatible material of the present invention, comprising: i) a substrate (1) comprising a biocompatible erodible fiber; (ii) a heparinized multilayer polymer layer-by-layer (LbL) coating (2) of the present invention; and an optional pre-coating deposit (3), wherein the multilayer polymer coating (2) comprises at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as an outer layer pair (21) forming a heparinized substrate for terminal functionalization of heparin (4), and the lower layer of the multilayer polymer coating (2) is formed by a layer pair of the same polymer. [Figure 3] Figure 1 shows the thickness (nm) of (CHI / HEP)n and (CHI / DS)n multilayer polymer coatings versus the LbL layer number (i.e., the number of layer pairs of anionic and cationic polymer layers deposited on a silicon wafer as described in Example 1). Note that a slope is obtained at higher layer numbers because the thickness of the first deposited layer is affected by the substrate. [Figure 4A] FIG. 1 shows the antithrombotic activity of various materials 1, 2, and 3 of the invention compared to comparative coatings C1-C6, expressed as anti-Xa values ​​(UI / ml) measured after 0 hours, 24 hours, 7 days, and 10 days of incubation. [Figure 4B] FIG. 1 shows the antithrombotic activity of various materials 1, 2, and 3 of the invention compared to comparative coatings C1-C6, expressed as anti-Xa values ​​(UI / ml) measured after 1, 2.5, 5, and 7 days of incubation. [Figure 4C] FIG. 1 shows the ability to inhibit thrombin generation (EPT: nmol / L / min) measured according to the assay of Example 2b after 0, 2.5 and 7 days of incubation. [Figure 5] FIG. 1 shows the antithrombotic activity of materials 4 and 5 of the invention, expressed as anti-Xa values ​​(UI / ml), measured after 1, 2.5, 5 and 7 days of incubation as described in Example 2. [Figure 6]4B and 4C show semiquantitative cell proliferation scores (0-4) for smooth muscle cells (SMC) (gray) and endothelial cells (EC) (black) from in vitro cell cultures tested on inventive materials 1 and 2 compared to comparative coatings C1 and C6 described in Example 2c after 5 days of incubation for the same samples shown in FIG. The higher the endothelial cell (EC) score on the cell-attracting coating, the better the endothelialization. DETAILED DESCRIPTION OF THE INVENTION

[0025] The term "heparin" refers to a heparin molecule, a fragment of a heparin molecule, or a heparin derivative. Heparin is a polysaccharide belonging to the glycosaminoglycan group with sulfonic acid groups distributed along the repeating unit. The heparin structure contains specific binding sequences that interact with blood proteins (e.g., thrombin (T) and antithrombin (AT), which catalyzes the inhibition of factor Xa). In an attempt to circumvent the problems of intravenously administered heparin, this molecule is most commonly immobilized on material surfaces by three different methods: ionic (electrostatic interactions), multipoint attachment (covalent bonding), and end-functionalization (covalent bonding). Attractive advantages of using this latter method include the orientation of the heparin molecule in the bulk and the retention of the active site responsible for inhibiting thrombin generation. Heparin derivatives can be any functional or structural variation of heparin. Heparin is attached to the final LbL layer of the multilayer polymer layer-by-layer (LbL) coating of the present invention.

[0026] The term "layer-by-layer (LbL) layer" defines a pair of anionic and cationic polymer layers. According to certain aspects, the number of LbL layers is from about 1 to about 8, typically from about 1 to about 5 (e.g., 4 or 5). According to more specific embodiments, for use as a biodegradable implant, it is desirable to keep the number of LbL layers in the material of the present invention as low as necessary to achieve the intended functionality. Additional layers increase the stiffness of the implant and also decrease its biodegradability. According to another specific embodiment, the multilayer polymer coating comprises a pair of alternating outer layer pairs of poly(allylamine hydrochloride) (PHA) and poly(sodium 4-styrenesulfonate) (PSS).

[0027] The term "nonwoven fibers" refers to any material construction that does not involve weaving or knitting of fibers. Nonwoven fibers can be nano- or micro-fibers or fibrils and materials, including nonwoven fibers obtained by various manufacturing procedures such as extrusion, expansion, or electrospinning / spraying.

[0028] The term "chitosan derivatives" refers to N-sulfofurfurylchitosan (SFC) and N-[(2-hydroxyl-3-trimethylammonium)propyl]chitosan chloride (HTACC) (Channasanon et al., 2007, J. Colloid Interface Sci., 316, pp. 331-343), N-glycidyltrimethylammonium chloride (Cui et al., 2010, Adv. Funct. Mater., 20, pp. 3303-3312); N-[(2-hydroxyl-3-trimethylammonium)propyl]chitosan chloride (HTACC), N-succinylchitosan (SCC) and N-sulfofurfurylchitosan (SFC) (Graisuwan, 2012, J. Colloid Interface Sci., 316, pp. 331-343). Sci., 376, pp. 177-188) or N-quaternized chitosan-like N,N,N-trimethylchitosan (TMC) (Martins, 2014, Int. J. Mol. Sci., 15, pp. 20800-20832).

[0029] The term "endothelial cell-attractive coating" refers to a coating that will allow cell attachment and induce rapid endothelialization due to a high affinity for endothelial cell growth on its surface. The endothelial cell-attractive properties of a coating can be determined by performing the assays described herein or any other suitable assay, such as a long-term preclinical in vivo implantation study. An example of a suitable endothelial cell-attractive coating according to the present invention includes at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as the outer layer pair of the coating.

[0030] The term "artificial device" includes artificial devices as defined herein that are intended to come into contact with blood (e.g., implantable or extracorporeal circulation devices, as well as catheters and other devices). Artificial devices include biodegradable and non-biodegradable devices. Implantable devices include artificial blood vessels, grafts, or shunts, cardiovascular patches, cardiovascular valves or leaflets, endoprostheses, such as stents, ventricular assist devices, artificial hearts, intravascular devices, such as filters, occlusion devices, and intravascular leads (pacemakers). Extracorporeal circulation devices include devices used for cardiopulmonary bypass, extracorporeal membrane oxygenation (ECMO), or other blood purification devices, such as dialysis, and their components.

[0031] The biodegradable device of the present invention may further comprise a mesh, preferably non-biodegradable, to improve mechanical stability during degradation of the biodegradable device; specifically, the mesh improves flexibility and provides anti-kinking properties to the implantable device, especially when inserted into a vascular graft. When used as a venous replacement, the incorporation of a mesh may prevent external compression of the blood vessel. Furthermore, in the case of a biodegradable vascular graft, aneurysm formation may be prevented later.

[0032] The term "biocompatible nonwoven fiber-containing substrate" includes materials suitable for implants, patches, or leaflets (e.g., degradable and non-degradable polymers, including but not limited to those described in this application).

[0033] The phrase "hemocompatible" refers to a material that can be used in contact with blood without causing harm (e.g., a material that is antithrombogenic). According to certain embodiments, hemocompatibility can be assessed by antithrombogenic effect over a period of at least one week or more (typically, from about one week to about one month) in a standard assay, such as those described herein.

[0034] As used herein, "treatment," "treating," and the like generally mean obtaining a desired pharmacological and / or physical and / or physiological effect. This effect may be prophylactic, in that it prevents or partially prevents a disease, symptom, or condition thereof, and / or therapeutic, in that it partially or completely cures a disease, condition, symptom, or adverse effects caused by a disease. The term "treatment," as used herein, covers any treatment of disease in a mammal (especially a human), including: (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., preventing the onset of the disease; or palliating the disease, i.e., causing regression of the disease and / or its symptoms or conditions (e.g., ameliorating or curing damage).

[0035] The term "subject," as used herein, refers to a mammal. For example, mammals contemplated by the present invention include humans, primates, and domestic animals, such as cattle, sheep, pigs, horses, particularly racehorses, laboratory rodents, and the like.

[0036] The term "effectiveness" of a treatment according to the present invention may be measured based on the course of disease changes in response to use according to the present invention. For example, the effectiveness of a device according to the present invention may be measured by the improvement of laboratory and clinical outcomes with an implanted device / material according to the present invention.

[0037] Materials according to the present invention Referring to the figures (particularly initially to FIG. 2A), a blood compatible material may be: - a support (1) comprising biocompatible nonwoven fibers; a heparinized multi-layer polymer layer-by-layer (LbL) coating (2) of said biocompatible nonwoven fiber, a heparinized multi-polymer layer-by-layer (LbL) coating (2), wherein the multi-polymer layer-by-layer (LbL) coating comprises at least one layer pair of an anionic polymer layer and a cationic polymer layer, and the multi-polymer layer-by-layer (LbL) coating is functionalized with heparin on the outer layer pair of the multi-polymer by terminal functionalization, wherein the anionic polymer is selected from heparin, poly(sodium 4-styrenesulfonate) (PSS), or a mixture thereof, and the cationic polymer is selected from chitosan or an analog thereof, and poly(allylamine hydrochloride) (PAH), or a mixture thereof; Includes:

[0038] In another further embodiment of the present invention, the material of the present invention further comprises an optional pre-coating deposit (3) on a support (1) comprising biocompatible nonwoven fibers, said pre-coating deposit comprising an adhesive for the heparinized multilayer polymer LbL coating (2). In certain embodiments, the adhesive can be a cationic polymer, such as poly(ethyleneimine) (PEI) or poly(lysine). Another cationic polymer that can be used as an adhesive can be poly(allylamine hydrochloride) (PAH).

[0039] In another further embodiment of the present invention, the material of the present invention further comprises an optional endothelial cell-attracting coating of a multi-layer polymer layer-by-layer (LbL) coating, wherein the endothelial cell-attracting coating is functionalized with heparin by terminal functionalization. According to certain aspects, the endothelial cell-attracting coating is particularly advantageous when used in combination with biodegradable nonwoven fibers.

[0040] In one particular embodiment, the support comprising biocompatible nonwoven fibers comprises or consists of biocompatible nonwoven fibers (eg, microfibers and / or nanofibers, or a combination thereof).

[0041] In another embodiment, the biocompatible nonwoven fibers have a diameter of 0.1 to 10 μm, for example, 0.5 to 5 μm or 1 to 5 μm.

[0042] In one particular embodiment, the nonwoven fibers form a three-dimensional network or matrix with a certain porosity defined by the interstices between the fibers.

[0043] Preferably, the nonwoven fibers are unoriented fibers, i.e., they do not have any particular orientation along a particular axis or plane, but can be aligned using various electrospinning parameters.

[0044] In one particular embodiment, said nonwoven fabric comprises or consists of several superimposed layers of fibers.

[0045] In a more particular embodiment, said nonwoven fabric comprises or consists of several superimposed layers with different porosities.

[0046] According to another particular embodiment, the nonwoven fibers according to the invention may be prepared as described in Pfeiffer et al., 2014, J. Biomed. Mater. Res., 102A, pp. 4500-4509.

[0047] In another embodiment, the biocompatible nonwoven fibers are biodegradable. Specifically, the biodegradable biocompatible nonwoven fibers may be found in biodegradable polymers selected from the group consisting of degradable polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly-L-lactide (PLLA), poly(glycerol sebacate) (PGS), polydioxanone (PDO, PDS), or poly-p-dioxanone, or PGA-poly-4-hydroxybutyrate (P4HB) copolymers, degradable polyurethane fibers, and combinations thereof, as well as any other polymers used in biomedical applications, for example, as described in Miranda et al., 2020, Antibiotics, 9, 174.

[0048] In a more particular embodiment, the biodegradable, biocompatible nonwoven fibers comprise or consist of polycaprolactone (PCL) fibers.

[0049] In a specific embodiment, the fibers are ε-PCL nanofibers, preferably electrospun ε-polycaprolactone nanofibers, which can be obtained, for example, according to the method described in Nottelet et al., 2009, J. Biomed. Mater. Res., 89A, 865-875.

[0050] In a specific embodiment, the fibers are polycaprolactones having a molecular weight in the range of 10 to 200 kDa, preferably in the range of 40 to 120 kDa, and preferably in the range of 60 to 100 kDa.

[0051] In one embodiment, the nonwoven comprises or consists of one or more layers with a porosity of 0.8 to 12 μm, which may be measured by the scaffold porosity (e) or void ratio as described in de Valence et al., 2012, Acta Biomater., 8, 3914-3920.

[0052] In another embodiment, the biocompatible nonwoven fibers are non-biodegradable. Specifically, non-biodegradable biocompatible nonwoven fibers can be found in polymers such as polyethylene terephthalate (PET), Dacron®, or expanded polytetrafluoroethylene (ePTFE) or polyurethane (PU), or others, which are widely used clinically as non-degradable synthetic vascular grafts.

[0053] In more specific embodiments, the non-biodegradable, biocompatible nonwoven fabric comprises or consists of ePTFE, for example, as described in Lian et al., 2003, J. Vasc. Surg., 37, 472-80.

[0054] In one embodiment, the multilayer polymer coating is a layer-by-layer (LbL) deposition multilayer coating. For example, an LbL deposition multilayer coating can be produced as described in Picart et al., 2015, Layer-by-Layer Films for Biomedical Applications, C. Picart, F. Caruso, and J.-C. Voegel (eds.), Wiley-VCH: Weinheim, Germany; or El-Khouri et al., 2011, Functional Polymeric Ultrathin Films, R. Advincula and W. Knoll (eds.), Wiley-VCH: Weinheim, Germany. Typically, the multilayer polymer coating has a total thickness of about 0.1 nm to about 500 nm, typically about 1 nm to about 500 nm, specifically 1 nm to about 100 nm, as measured, for example, by ellipsometry on a silicon wafer, as shown in FIG. 3, with each layer pair having a thickness of about 0.2 nm to about 50 nm (e.g., about 1 to 25 nm).

[0055] In one embodiment, the anionic or cationic polymer has a molecular weight

[0056]

number

[0057] is in the range of 5 kDa to 1 MDa, for example 7.5 kDa to about 1000 kDa, for example about 5 to 200 kDa, for example about 5 to 75 kDa (e.g., about 7.5 kDa), or about 15 kDa to 75 kDa. According to certain embodiments, suitable anionic or cationic polymers according to the present invention have a molecular weight of less than 75 kDa.

[0058] In one particular embodiment, the anionic or cationic polymer has a molecular weight

[0059]

number

[0060] is in the range of 7.5kDa to 75kDa.

[0061] In one embodiment, the anionic polymer is selected from heparin, and poly(sodium 4-styrenesulfonate) (PSS), or a mixture thereof.

[0062] In another embodiment, the cationic polymer is selected from chitosan or an analog thereof, and poly(allylamine hydrochloride) (PAH), or a mixture thereof.

[0063] In a further embodiment, the anionic polymer is selected from heparin and poly(sodium 4-styrenesulfonate) (PSS), and the cationic polymer is selected from chitosan or an analog thereof, and poly(allylamine hydrochloride) (PAH).

[0064] In one particular embodiment, the anionic polymer is heparin.

[0065] In one particular embodiment, the anionic polymer is poly(sodium 4-styrenesulfonate) (PSS).

[0066] In one particular embodiment, the cationic polymer is chitosan or an analog thereof (eg, an N-alkylated chitosan, such as trimethylchitosan).

[0067] In one particular embodiment, the chitosan has a molecular weight of about 7.5 kDa to 75 kDa.

[0068] In another particular embodiment, the chitosan has a degree of deacetylation of at least 80%, preferably at least 85%, and typically from about 80 to about 95%.

[0069] In another particular embodiment, trimethylchitosan is prepared as described in Mourya et al., 2009, J. Mater. Sci. Mater. Med., 20, 1057-79, or Malik et al., 2018, Int. J. Nanomedicine, 13, 7959-7970, or Kulkarni et al., 2017, Carbohydr. Polym., 157, 875-902.

[0070] In one particular embodiment, the anionic polymer is poly(allylamine hydrochloride) (PAH).

[0071] In a more specific embodiment, the multilayer polymer LbL coating comprises from about 3 layer pairs to about 8 layer pairs of anionic and cationic polymer layers according to the present invention, the layer pairs being arranged such that the anionic and cationic polymer layers alternate in the multilayer polymer coating.

[0072] In a more specific embodiment, the multilayer polymer LbL coating of the material of the present invention comprises at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as the outer pair of layers of said coating, which are functionalized with terminally functionalized heparin.

[0073] According to more specific embodiments, the multilayer polymer LbL coating comprises about 1 layer pair to about 8 layer pairs of anionic and cationic polymer layers, wherein the anionic polymer is heparin, the cationic polymer layer is selected from chitosan or analogs thereof, and the outer layer pair of the coating is an alternating layer pair of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) functionalized with end-functionalized heparin.

[0074] End-functionalization of the membrane with heparin is achieved by reaction of heparin with amine groups in the outer layer of the multi-polymer layer-by-layer coating.

[0075] In one particular embodiment, a substrate comprising biocompatible nonwoven fibers may first be pre-coated with a deposit comprising an adhesive for a multi-layer polymer coating.

[0076] In certain embodiments, the adhesive is a cationic polymer containing multiple primary or secondary amino groups (eg, poly(ethyleneimine) (PEI)).

[0077] Referring to the figures (particularly initially to Figure 2), a heparinized multilayer polymer LbL coating (2) is obtained by functionalizing heparin (4) onto the outer layer of said multilayer polymer coating (2) by terminal functionalization, independent of the fact that heparin can be used as the anionic polymer in said multilayer polymer LbL coating (2).

[0078] Various end-functionalization strategies for heparin are known, as described, for example, in Biran et al., 2016, Adv. Drug Deliv. Rev., 112, 12-23.

[0079] According to a more specific embodiment, heparin is functionalized on the outer layer pair of the multilayer polymeric LbL coating via end-attachment (EPA), where the reducing end of the heparin (typically first partially depolymerized by nitrous acid deamination) is covalently bound to the amino functional group of the final layer of the multilayer polymeric LbL coating by reductive amination.

[0080] According to further particular embodiments, heparin is functionalized on the outer layer pair of said multilayer polymeric LbL coating via terminal conjugation (EPC), in particular heparin is bound to the amino functional groups of the final layer of the multilayer polymeric LbL coating, as described, for example, in Hsieh et al., 2017, supra.

[0081] According to a more specific embodiment, the heparin is functionalized after diazotization of the heparin to provide highly reactive aldehyde groups on the heparin prior to functionalization onto the polymer coating.

[0082] According to further particular embodiments, the terminal conjugation of heparin can provide sufficient access for endothelial cells to the outer layer of the LbL coating, particularly the cell-attractive coating of a multilayer polymer layer-by-layer (LbL) coating functionalized with heparin by terminal conjugation.

[0083] Referring initially to the figures (particularly Figures 2B and 2C), in another specific embodiment, a multilayer polymer LbL coating on a substrate comprising biocompatible nonwoven fibers includes at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as the outer layer pair (21), forming a heparinized substrate for end-functionalization of heparin (4). In this case, heparin (4) is end-functionalized to the multilayer polymer coating (2), with the underlying layer of the multilayer polymer coating being formed from a layer pair of either the same polymer (Figure 2C) or, preferably, another polymer described herein ((22) in Figure 2B).

[0084] According to a more specific embodiment, a multilayer polymer LbL coating of a substrate comprising biocompatible nonwoven fibers is achieved by first coating the substrate (optionally pre-coated with a pre-coating agent) with an anionic polymer of a first LbL layer pair.

[0085] In another particular embodiment, the multilayer polymer LbL coating of the substrate comprises several layer pairs of anionic and cationic polymer layers, where the anionic polymer layers can be formed of the same or different anionic or cationic polymers between the various layer pairs.

[0086] In another particular embodiment, the multilayer polymer LbL coating of the substrate comprises about 3 to 8 (particularly 3 to 5) alternating anionic / cationic polymer layer pairs, which polymer layer pairs are selected from heparin / chitosan or analogue layer pairs, and PAH / PSS layer pairs, or combinations thereof.

[0087] In another particular embodiment, the multilayer polymer coating of the implantable material support comprises about 3 to 8 (e.g., about 3 to 7, e.g., about 3 to about 5, particularly 4 to 5) layer pairs of anionic and cationic polymer layers, wherein the anionic polymer is heparin and the cationic polymer is chitosan or an analog thereof.

[0088] In another particular embodiment, the multilayer polymer LbL coating of the substrate comprises about 3 to 8 (e.g., about 3 to 7, e.g., about 3 to about 4, particularly 4 to 5) layer pairs of anionic and cationic polymer layers, wherein the anionic polymer is PSS and the cationic polymer is PAH.

[0089] In another specific embodiment, the multilayer polymer LbL coating of the substrate comprises about 3 to about 7 (e.g., about 3 to about 5) layer pairs (e.g., 3, 4, or 5) of anionic polymer layers and cationic polymer layers, wherein the anionic polymer is heparin (HEP) and the cationic polymer is chitosan (CHI), the outer polymer layer pairs consist of about 1 to about 2 layer pairs of anionic polymer layers and cationic polymer layers, wherein the anionic polymer is PSS and the cationic polymer is PAH, and the outer polymer layer pairs PSS / PAH are end-functionalized with heparin.

[0090] In one particular embodiment, in a multilayer polymer coating comprising or consisting of a (HEP / CH) layer pair and a multilayer polymer coating comprising or consisting of a (PSS / PAH) layer pair, the thickness increment per layer pair from the multilayer polymer LbL is in the range of 1 to 20 nm.

[0091] According to an advantageous aspect of the present invention, the thickness of the multi-polymer LbL coating can be adjusted by selecting specific polymer layer pairs.

[0092] In another more particular embodiment, the material according to the invention is selected from the group consisting of: - (optional adhesive)-(Hep / Chi)5-Hep, where the last LbL layer of (Hep / Chi)5 is functionalized with heparin by terminal conjugation; and - (Optional Adhesive)-(PSS / PAH)-Hep, where the last LbL layer of -(PSS / PAH) is functionalized with heparin by terminal conjugation. The present invention relates to a method for producing a biocompatible nonwoven fiber coated with a system comprising a multilayer (LbL) polymer assembly from

[0093] According to a particular embodiment, an adhesive is present.

[0094] According to another particular embodiment, the adhesive is PEI.

[0095] According to another particular embodiment, the adhesive is a PAH.

[0096] Preparation of the material according to the present invention According to a particular embodiment, - providing a biocompatible nonwoven fabric as a support; - coating said substrate with a multilayered polymeric LbL coating, a coating step in which the multilayer polymer LbL coating comprises at least one layer pair of an anionic polymer layer and a cationic polymer layer, wherein the anionic polymer is selected from heparin, poly(sodium 4-styrenesulfonate) (PSS), or a mixture thereof, and the cationic polymer is selected from chitosan or a mixture thereof, and poly(allylamine hydrochloride) (PAH), or a mixture thereof; - functionalizing the outer layer pair of the multilayer polymer layer-by-layer (LbL) coating with heparin by terminal functionalization. A method for preparing a blood compatible material is provided, comprising:

[0097] According to certain embodiments, the implantable material is in the form of a biocompatible nonwoven fiber that is provided as a support by electrospinning.

[0098] According to another particular embodiment, the biocompatible nonwoven fibers are spun to form a support in the form of a tubular structure that defines a lumen, the wall of which forms the luminal surface.

[0099] According to certain embodiments, the nonwoven fiber substrate is pretreated by immersion in an alcohol solution for about 10 minutes, followed by immersion in ultrapure water for about 10 minutes, and then drying before coating (e.g., in a solution containing ethanol, such as about 100% to about 95% ethanol).

[0100] According to another particular embodiment, the method for preparing a blood compatible material further comprises an optional pre-coating step of providing a pre-coating deposit of adhesive on the surface of the implantable material prior to the multi-layered polymer coating.

[0101] According to certain embodiments, an adhesive pre-coating step for multi-layered polymer LbL coatings is advantageously used when the first polymer layer of the first layer pair is a cationic polymer.

[0102] According to another particular embodiment, a pre-coating step with adhesive for the multi-layered polymer coating is advantageous when the implantable material is PCL.

[0103] According to another particular embodiment, there is provided a method for preparing a blood compatible material according to the present invention, wherein a multilayered polymer LbL coating is applied to a biocompatible nonwoven fiber (either directly or onto a pre-coated biocompatible nonwoven fiber) by layer-by-layer (LbL) deposition.

[0104] According to another particular embodiment, there is provided a method for preparing a blood compatible material according to the present invention, wherein heparin is functionalized to a multilayered polymer coating by terminal functionalization.

[0105] According to another specific embodiment, there is provided a method for preparing a blood compatible material according to the present invention, wherein the multilayer polymeric LbL coating of a biocompatible nonwoven fiber comprises at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as an outer layer pair, forming a heparinized substrate for end-functionalization with heparin.

[0106] According to another specific embodiment, the heparinized multilayer polymer coating of the present invention has antithrombogenic and / or cell proliferation properties and can therefore be used on any substrate intended to come into contact with blood for any in vitro, animal, or human application.

[0107] The artificial device of the present invention According to another particular embodiment, the substrate of the material of the present invention may be an implantable or non-implantable medical device in contact with blood, in particular a stable or biodegradable implantable prosthetic device in contact with blood, such as those defined herein.

[0108] According to certain embodiments, the implantable medical devices of the present invention have antithrombotic properties.

[0109] According to certain embodiments, the implantable medical device of the present invention has anticoagulant properties.

[0110] According to certain embodiments, the implantable medical devices of the present invention do not require a pre-endothelialization step prior to implantation.

[0111] According to certain embodiments, there is provided an implantable medical device suitable for contact with blood, said device comprising a surface (e.g., a wall) comprising a biocompatible nonwoven fiber, said surface being coated with a multi-layer polymeric coating according to the present invention.

[0112] According to certain embodiments, an implantable medical device suitable for contact with blood is provided, wherein the surface comprising biodegradable and biocompatible nonwoven fibers is formed from electrospun biocompatible nonwoven fibers.

[0113] In one embodiment, a biodegradable implantable medical device, particularly a vascular graft, is provided having a tubular structure that defines a lumen, with the wall of the device forming the luminal surface.

[0114] According to certain embodiments, an implantable medical device suitable for contact with blood is provided that is a vascular graft.

[0115] In one embodiment, the device is a biodegradable vascular graft having a tubular structure that forms a lumen, with the wall of the device forming the luminal surface, and the biodegradable vascular graft can be of any size, diameter, or configuration (straight or branched), but particularly has an inner diameter of less than 6 mm (ID<6 mm), typically from about 1 to about 6 mm.

[0116] In one specific embodiment, the biodegradable vascular graft is a coronary vascular graft.

[0117] Advantageously, implantable prosthetic devices, such as biodegradable vascular grafts according to the present invention, are permanent or temporary intracorporeal medical devices.

[0118] In one embodiment, the implantable prosthetic device of the present invention is selected from the group consisting of a stent, e.g., a bifurcated stent, a balloon-expandable stent, a self-expanding stent, a stent-graft, e.g., a bifurcated stent-graft, a graft, e.g., a vascular graft, a bifurcated graft, an embolic filter, a vascular prosthesis, a drug delivery device / balloon, a patch, an intravascular occlusion device, a CNS shunt (e.g., a ventriculopleural shunt, a VA shunt, or a VP shunt), a ventriculoperitoneal shunt, a ventriculoatrial shunt, a portovenous shunt, and a shunt for ascites, a heart valve leaflet, a shunt for pediatric cardiac surgery, a vascular graft, and a shunt for dialysis access surgery. Furthermore, the biodegradable implantable medical device of the present invention is also an extracorporeal cardiac assist device such as an artificial heart, a left ventricular assist device (LVAD), or a non-implantable extracorporeal assist device such as cardiopulmonary bypass (CPB), extracorporeal membrane oxygenation (ECMO), or a blood purification device, and a catheter therefor.

[0119] According to a further specific embodiment, there is provided an extracorporeal circulation device, such as a cardiopulmonary bypass / ECMO system, including an oxygenator, or a hemodialysis / filtration system, or a blood purification system, as well as all catheters and tubes, comprising the material according to the present invention (particularly coating material 1 or 3 according to the present invention) or a material obtainable according to the method of the present invention.

[0120] Preparation of the Prosthetic Device of the Invention According to a particular embodiment, - providing an implantable device or a part thereof (e.g., a wall of the implantable device) comprising a biocompatible nonwoven fabric as a support; - coating said substrate with a multilayered polymeric LbL coating according to the invention A method for preparing an implantable device is provided, comprising:

[0121] According to a particular embodiment, - forming the wall by either electrospinning or extrusion / expansion of biocompatible nonwoven fibers; - coating said wall with a multilayer polymer coating by layer-by-layer (LbL) deposition, the multilayer polymer coating comprising at least one layer pair of an anionic polymer layer and a cationic polymer layer according to the invention; - functionalizing said multilayered polymeric LbL coating with heparin by terminal functionalization on its outer layer; A method for preparing a blood compatible implantable device is provided, comprising:

[0122] According to certain embodiments, the implantable device or a portion thereof is provided as a support by forming a wall by electrospinning of biocompatible nonwoven fibers.

[0123] According to a specific embodiment, a method for preparing an implantable device of the present invention is provided, in which a non-degradable or degradable mesh is introduced into a non-biodegradable or fully biodegradable implantable device (e.g., prosthetic) that comes into contact with blood, in particular a fully biodegradable vascular graft. According to this specific embodiment, when the fully biodegradable vascular graft is implanted in the arterial or venous circulation or any other tubular structure, the non-degradable or degradable mesh prevents the graft wall from rupturing, expanding, or kinking in the event of insufficient revascularization after complete polymer degradation. The mesh improves the mechanical stability of the graft before, during, and after degradation. Thus, the present invention relates to a prosthetic device comprising a fully biodegradable implantable prosthetic device as defined herein and a non-degradable or degradable mesh.

[0124] patient In one embodiment, a patient of the present invention is a subject suffering from or at risk of suffering from ischemic, degenerative, or congenital cardiovascular disease, trauma (e.g., neurological, peripheral, cardiac, vascular, or orthopedic trauma), or organ loss or failure, such as end-stage renal disease (ESRD).

[0125] In a further embodiment, a patient according to the present invention is a subject undergoing reconstructive surgery, such as revascularization surgery.

[0126] Use according to the present invention According to certain embodiments, the materials, processes, and methods of the present invention are useful for surgical or therapeutic treatment of cardiovascular disease, particularly cardiovascular ischemic disease, e.g., coronary artery disease, peripheral vascular disease, or access surgery for dialysis, or for graft or shunt applications in pediatric patients, pediatric cardiovascular malformations, trauma, or repair surgery.

[0127] According to certain aspects, the biocompatible materials of the present invention may be useful for the preparation of non-degradable or degradable implantable prosthetic devices (e.g., vascular grafts (endoprostheses) useful for the surgical or therapeutic treatment of humans or animals, in particular cardiovascular disease, in particular cardiovascular ischemic disease, e.g., coronary artery disease, or peripheral vascular disease, or access surgery for dialysis patients, pediatric cardiovascular malformations, trauma, and repair surgery).

[0128] Some of the problems associated with the material properties of non-degradable polymers such as ePTFE, which is extremely hydrophobic (contact angle greater than 100°), may be overcome by the proposed material in this invention, as it renders the surface more hydrophilic, thereby promoting cell adhesion / growth and less thrombogenicity, and therefore more biocompatible, and may also reduce inflammation / foreign body reaction, potentially leading to a reduction in late complications such as intimal hyperplasia and calcification.

[0129] An illustrative example of the invention will now be described in more detail and with reference to embodiments shown in the drawings. [Example]

[0130] Example 1: Preparation of vascular grafts using materials of the present invention The material of the present invention was applied onto a substrate useful as an implantable vascular graft.

[0131] All reagents were used as received without any additional purification steps.

[0132] Poly(allylamine hydrochloride) (PAH, ≈50,000 g / mol), poly(sodium 4-styrenesulfonate) (PSS, ≈70,000 g / mol), and dextran sulfate sodium salt (DS, ≥500,000 g / mol) from Leuconostoc ssp. were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Poly(L-lysine) hydrobromide (PLL, ≈15,000–30,000 g / mol) and chitosan (CHI, low molecular weight, 75–85% deacetylated) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Branched poly(ethyleneimine) (PEI, Lupasol WF, ≈25,000 g / mol) was purchased from BASF (Ludwigshafen, Germany). Heparin sodium salt (>180 USP units / mg, reference H4784 from SIGMA) derived from porcine intestinal mucosa was used. N,N,N-trimethylchitosan (TMC,

[0133]

number

[0134] ≈90,000 g / mol, degree of quaternization 0.25) was obtained by chemical modification of chitosan (Golden-Shell Biochemical Co., China) with methyl iodide (CHI) as described elsewhere (Verheul et al. 2008 Biomaterials, 29, 3642–3649; Martins et al. 2013 Carbohydr. Res., 381, 153–160).

[0135] A solution of PEI was prepared in ultrapure water at a concentration of 2.5 mg / mL. The polyelectrolyte solutions used for the assembly of LbL films can be classified into three different groups based on the polyanion used: HEP, DS, or PSS.

[0136] For HEP-containing LbL films, polymer solutions were prepared in 0.15 M sodium chloride solution at a concentration of 1.0 mg / mL, except for CHI. Chitosan was dissolved in a solution of acetic acid (0.5 M) and 0.15 M NaCl to a final concentration of 2.0 mg / mL. A HEP solution at 1 mg / mL was prepared using acetate buffer (pH 4).

[0137] For DS-based LbL films, an aqueous solution of DS (pH ≈ 6.5) was prepared in 1 M NaCl at a polymer concentration of 1 mg / mL. Chitosan was dissolved in a solution of acetic acid (0.5 M) and 1 M NaCl to a final concentration of 2.0 mg / mL. A PLL solution was prepared at a concentration of 1 mg / mL and 1 M NaCl.

[0138] For PSS-containing LbL films, polymer solutions were prepared in 0.15 M sodium chloride solution at a concentration of 1.0 mg / mL.

[0139] No final adjustment of the pH value was made to any of the polyelectrolyte solutions.

[0140] A mixture of DS:HEP 1:10 was prepared in acetate buffer at 0.15 M NaCl, taking into account the molar ratio (number of chains) of each component per milliliter, maintaining a final polymer concentration of 1 mg / mL (77.3 mg of DS and 23.2 mg of HEP dissolved in 100 mL of buffer).

[0141] a) providing a support comprising biocompatible nonwoven fibers; Biocompatible nonwoven fibers were used as a support for the coating of the present invention. For this purpose, ε-polycaprolactone (PCL) micro- and nanofibrous scaffolds were prepared by electrospinning. Briefly, a solution of 15% PCL (80,000 Da, Sigma) in CHCl3 / EtOH (70% v / v) was charged at 20 kV (Spellman High Voltage Electronics Ltd, Pulborough, West Sussex, UK) and pumped through a stainless steel needle at a continuous rate of 12 mL / h by a syringe pump (Fresenius Vial SA, Brezins, France). The polymer fiber was then transferred to a grounded, rotating (4,500 rpm) and translating (200 travel s) tube mounted on a home-built stand. -1 The PCL fibers were harvested by a stainless steel mandrel (2 mm diameter, or less than 2 m, or more than 2 m) with a needle-to-recovery distance of 20 cm (and 4 cm amplitude). After electrospinning, the grafts were placed in vacuum overnight to remove any residual solvent. The average size and distribution of such PCL fibers was measured in a previous study, corresponding to an average fiber diameter of 2.2 ± 0.6 μm.

[0142] The resulting fiber meshes (referred to herein as "grafts" and "patches") were used as bioartificial surfaces mimicking vascular grafts used in cardiovascular surgical implants. Brief pretreatment of the nonwoven fiber supports (e.g., grafts / patches) was performed by immersion in ethanol for 10 minutes and rinsing with ultrapure water for 10 minutes, followed by drying. Sterilization prior to implantation was performed using gamma radiation with a minimum dose of 25 kGy and a maximum dose of 32 kGy.

[0143] b) optionally coating the substrate with a pre-coating deposit comprising an adhesive; If a pre-coating deposit of adhesive is used, the PEI solution is coated onto the nonwoven fiber substrate obtained above by immersion for 15 minutes and rinsing with ultrapure water three times for 2 minutes, followed by drying.

[0144] c) Coating a substrate (pre-coated with an adhesive or not) with a multi-layer polymer LbL coating comprising at least one layer pair of an anionic polymer layer and a cationic polymer layer of the present invention. Approximately 3 cm x 6 cm samples of the PCL-patch obtained above were coated by immersing the substrate in the solution. The duration of the polymer deposition and rinsing steps was approximately 1 min in the polyelectrolyte solution, followed by 3 x 8 s rinses with ultrapure water.

[0145] The following materials of the present invention, including LbL film combinations of the present invention (LbL Systems 1-3), and comparative coatings C1-C6, were prepared on biocompatible nonwoven fiber surfaces of various devices, as listed in Table 1 below.

[0146] [Table 1]

[0147] d) Heparinization of the multilayered polymer coating on the final LbL layer A heparinized multi-layer polymeric LbL coating is obtained by functionalizing heparin onto the outer layer of said multi-layer polymeric coating, regardless of the fact that heparin is incorporated as an anionic polymer of said multi-layer polymeric LbL coating or not.

[0148] Heparinization can be carried out by a variety of known techniques. In certain embodiments, heparin molecules are attached to the surface of the multilayered polymer coating using terminal conjugation (EPC), as described in Hsieh et al., 2017, supra.

[0149] For heparin terminal conjugation, the material obtained in the previous step was immersed in Hep EPC solution (0.02 M monobasic sodium phosphate, 0.2 M sodium chloride, and 3 mg mL) for 24 h. -1Immerse in sodium cyanoborohydride (C1H2O), agitate using an orbital shaker (200 rpm), then rinse with ultrapure water 3 x 2 min and dry.

[0150] According to certain embodiments, heparinized substrates advantageous for heparin terminal conjugation are formed with at least one outer layer pair comprising poly(allylamine hydrochloride) (PAH) and poly(allylamine hydrochloride) (PSS).

[0151] The amount of heparin deposited on the multilayered polymer coating was determined using the carbazole assay described in US Pat. No. 4,613,665 to be approximately 9.6 μg / cm 2 .

[0152] These results suggest that the multilayered polymer LbL coating presents a sufficient amount of primary amino groups in the bulk direction to react efficiently with heparin (aldehyde-terminated) molecules.

[0153] Given the current clinical use of vascular grafts and patches made of microporous, non-degradable polymers such as ePTFE, the coatings of the present invention have also been tested on these materials. To this end, surfaces prepared with and without conventional surface activation procedures (e.g., plasma treatment, etc.) for ePTFE and layer-by-layer coating methods were compared with those used for PCL as described above, as follows:

[0154] a) providing a support comprising biocompatible nonwoven fibers; Prior to deposition, the e-PTFE patches were soaked in EtOH for 1 hour and then in Milli-Q water for 30 minutes.

[0155] b) optionally coating the substrate with a pre-coating deposit comprising an adhesive; When adhesive pre-coating deposits were used, the patches were immersed in the PEI or PAH solution for 20 minutes, followed by three 2-minute rinses with Milli-Q water.

[0156] c) Coating a substrate (pre-coated with an adhesive or not) with a multi-layer polymer LbL coating comprising at least one layer pair of an anionic polymer layer and a cationic polymer layer of the present invention. The ePTFE-patch obtained above was coated by deposition of a (PSS / PAH)4 multilayer film by immersing the patch in the corresponding polyelectrolyte (PSS or PAH) for 1 min, followed by rinsing three times with Milli-Q water for 8 s.

[0157] The following materials of the present invention (LbL Systems 4-5) comprising LbL film combinations of the present invention are prepared on biocompatible nonwoven fiber surfaces of various devices, as listed in Table 2 below.

[0158] [Table 2]

[0159] d) Heparinization of the multilayered polymer coating on the final LbL layer A heparinized multilayer polymer LbL coating was obtained by functionalizing the outer layer of the LbL-modified e-PTFE patch obtained above with the EPC procedure described above for the PCL sample by end-attaching heparin. The e-PTFE patch was dried only at the end of the deposition process, rather than after each deposition step as in the case of the PCL sample.

[0160] Example 2: Characterization of implants coated with materials of the present invention The implants obtained in Example 1 were tested as follows.

[0161] a) Graft stability The stability of such films was evaluated under conditions similar to those used in antithrombotic experiments, using simulated immersion in 0.15 M NaCl aqueous solution at room temperature or 37°C for at least 24 hours and up to 7 and / or 10 days, taking into account additional events that would primarily involve protein adsorption on top of the coating. Total film thickness (nm) over time (h) was assessed by ellipsometry on silicon wafers, and the multilayered polymer coating thickness maintained more than 90% of its initial value, suggesting that film integrity was maintained for more than 7 days under these conditions.

[0162] This stability suggests that it may help the host body to undergo the endothelialization process safely from thrombus formation and the complications that result therefrom.

[0163] Furthermore, we observed that gamma sterilization (25 KGy) and storage (refrigerated for up to 9 months) did not alter the stability results for the vascular grafts.

[0164] b) Antithrombotic properties The antithrombotic performance of the vascular grafts was evaluated in vitro using two complementary methods.

[0165] First, the amount of anticoagulant released from the grafts when incubated with human plasma was evaluated. To this end, grafts were placed in tubes and incubated with 700 μL of pooled human plasma (CRYOcheck, PrecisionBiologic) under static conditions at 37°C for 24 hours, 60 hours, 5 days, 7 days, and 10 days, in triplicate for each condition. Anti-Xa activity in the plasma after each incubation period was measured with a chromogenic anti-Xa assay using an Atellica instrument (Siemens, Germany). Next, the efficacy of each graft in inhibiting thrombin formation in pooled human plasma (CRYOcheck, PrecisionBiologic) was evaluated using a Calibrated Automated Thrombogram® (Stago, Asnieres-sur-Seine, France). To this end, grafts were incubated as described above and then placed in wells with 300 μL of fresh pooled plasma (CRYOcheck). Thrombin generation was then measured with the PPP reagent (Stago, reference 86193) at 37° C. as recommended by the manufacturer.

[0166] The results for implants coated with materials of the present invention compared to comparison systems C1-C6 are shown in Figures 4A-B and 4C, respectively. Control patches without heparin show no anti-Xa increase. The longest anti-Xa effect was observed with PEI-(Hep / Chi)5-Hep terminal (1). The variability in the results for the present invention (Coating 1) between Figures 4A and 4B is due to different time points and different series of experiments.

[0167] FIG. 4A shows that the coating using the chitosan analog TMC (Inventive Coating 3) had similar anticoagulant properties as the one with chitosan (Inventive Coating 1).

[0168] The (PSS / PAH)-based coatings of the present invention (Inventive Coatings 4 and 5) were also applied to ePTFE patches and analyzed as described above. Figure 5 shows that the (PSS / PAH)-based coatings also possessed anticoagulant properties on ePTFE, and this property was not dependent on the presence of a PEI sublayer as an adhesive.

[0169] From these data it can be clearly seen that the materials according to the invention show sustained inhibition of thrombin in human plasma for up to 5 or 7 days compared to controls in which thrombin is generated normally at all time points. Notably, the inhibition of thrombin generation, as assessed by anti-Xa assay in supernatant plasma, was effective in all samples according to the invention despite an initial release of anticoagulant (heparin) during the first 24 hours of incubation.

[0170] c) Cell adhesion The performance of the vascular grafts in recruiting endothelial cells and smooth muscle cells was assessed in an in vitro cell proliferation assay described below.

[0171] Cell numbers were semiquantitatively counted after 5 days of incubation with smooth muscle cells (SMCs) and endothelial cells (ECs), as shown in Figure 6. SMC proliferation is important in degradable materials to repopulate the vessel wall and trigger ECM formation. Heparin is known to reduce this activity, as shown in Figure 6 for LbL system 1.

[0172] These results confirm that endothelial cells can rapidly attach to multilayer polymeric LbL coatings when the coatings contain a heparinized substrate comprising at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as the outer layer pair, and when heparin is functionalized by terminal conjugation to the heparinized substrate.

[0173] This finding confirms that rapid and complete endothelialization can be obtained with the material of the present invention, which is particularly useful in the context of biodegradable vascular grafts comprising the material of the present invention whose surface is in contact with blood, which can induce in vivo conversion to neovessels.

[0174] Taken together, these data support the ability of the materials of the present invention to inhibit clotting and thrombus formation after implantation of biocompatible devices that come into contact with blood, particularly, but not exclusively, vascular grafts.

[0175] Furthermore, the method for preparing the material of the present invention advantageously provides a method for obtaining biocompatible nonwoven fibers that are uniformly coated with a multilayered polymer coating by LbL techniques, the uniformity being evidenced not only by optical and fluorescent microscopy but also primarily by the presence of a strong anticoagulant response that confirms that essentially all of the fibers are coated, thereby preventing clotting events at micro-defects.

[0176] LbL coating of the material (2 of the present invention) further allowed cell adhesion and proliferation, particularly of endothelial cells (Figure 6). Finally, terminal functionalization (particularly heparin conjugation) significantly improved the performance of the device (patch) of the present invention in terms of delaying prethrombin formation and consequent thrombus formation.

Claims

1. - a support comprising biocompatible nonwoven fibers; a heparinized multi-layer polymer layer-by-layer (LbL) coating on the biocompatible nonwoven fiber, the multi-layer polymer coating comprising at least one layer pair of an anionic polymer layer and a cationic polymer layer, the multi-layer polymer layer-by-layer (LbL) coating being functionalized with heparin on an outer layer pair of the multi-layer polymer coating by terminal functionalization, the anionic polymer being selected from heparin, poly(sodium 4-styrenesulfonate) (PSS), or a mixture thereof, the cationic polymer being selected from chitosan or an analog thereof, and poly(allylamine hydrochloride) (PAH), or a mixture thereof, and the chitosan analog being N-alkylated chitosan; blood compatible materials, including

2. 10. The material of claim 1, wherein the biocompatible nonwoven fiber is biodegradable.

3. 3. The material of claim 1 or 2, wherein the biocompatible nonwoven fibers are comprised of a biodegradable polymer selected from the group consisting of degradable polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly-L-lactide (PLLA), poly(glycerol sebacate) (PGS), polydioxanone (PDO, PDS), or poly-p-dioxanone, or PGA-poly-4-hydroxybutyrate (P4HB) copolymers, degradable polyurethane fibers, and combinations thereof.

4. 4. The material according to claim 1, wherein the biodegradable, biocompatible nonwoven fibers comprise or consist solely of polycaprolactone (PCL) fibers.

5. A material described in any one of claims 1 to 4, wherein the biodegradable, biocompatible nonwoven fiber comprises or consists solely of electrospun ε-PCL nano / microfibers.

6. 10. The material of claim 1, wherein the biocompatible nonwoven fiber is non-biodegradable.

7. 10. The material of claim 1 or 6, wherein the biocompatible nonwoven fabric is made of a non-biodegradable polymer selected from polyethylene terephthalate (PET), Dacron®, and expanded polytetrafluoroethylene (ePTFE) or polyurethane (PU).

8. 8. The material of any one of claims 1 to 7, wherein the biocompatible nonwoven fibers have a diameter of about 0.1 to 10 μm.

9. A material described in any one of claims 1 to 8, wherein the biocompatible nonwoven fibers have a diameter of approximately 0.5 to 5 μm.

10. 9. The material of claim 1, wherein the anionic polymer is heparin.

11. 9. The material according to claim 1, wherein the cationic polymer is chitosan or an analog thereof, and the chitosan analog is N-alkylated chitosan.

12. 12. The material of claim 1, further comprising an endothelial cell-attracting coating of the multilayer polymer layer-by-layer (LbL) coating, wherein heparin is functionalized on the endothelial cell-attracting coating by terminal functionalization.

13. 13. The material of any one of claims 1 to 12, wherein the biocompatible nonwoven fiber is first coated with a pre-coating deposit comprising an adhesive for the multi-layer polymer coating.

14. The material of claim 13, wherein the adhesive for the multi-layer polymer coating is a cationic polymer.

15. The material of claim 13 or 14, wherein the adhesive for the multilayer polymer coating is selected from poly(ethyleneimine) (PEI) and poly(allylamine hydrochloride) (PAH).

16. 16. The material of any one of claims 1 to 15, wherein the multilayer polymer LbL coating comprises 3 to 8 layer pairs of anionic and cationic polymer layers as defined in any one of claims 1 to 15, the layer pairs being arranged such that the anionic polymer and the cationic polymer layers alternate in the multilayer polymer coating.

17. 16. The material of any one of claims 1 to 15, wherein the multilayer polymer LbL coating comprises 3 to 8 alternating anionic / cationic polymer layer pairs, the polymer layer pairs being selected from heparin / chitosan or analogue layer pairs, and PAH / PSS layer pairs, or combinations thereof.

18. 16. The material of any one of claims 1 to 15, wherein the multilayer polymer coating of the biocompatible nonwoven fiber comprises at least one pair of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) as an outer pair of layers, forming a substrate for end-functionalization of heparin.

19. The following groups: - (Optional adhesive) - (Hep / Chi) 5 -Hep(in the formula, (Hep / Chi) 5 the final LbL layer is functionalized with heparin by terminal conjugation); and - (Optional adhesive) - (PSS / PAH) 4 -Hep(in the formula, (PSS / PAH) 4 The final LbL layer is functionalized with heparin by terminal conjugation.

19. The material of any one of claims 1 to 18, comprising a biocompatible nonwoven fiber coated with a system comprising a multilayer LbL polymer assembly from

20. - providing a biocompatible nonwoven fabric as a support; - optionally coating said support with a pre-coating deposit comprising an adhesive for a multi-layered polymer LbL coating; - coating the pre-coated or non-pre-coated substrate with a multilayered polymeric LbL coating, the multilayered polymeric LbL coating comprising at least one layer pair of an anionic polymer layer and a cationic polymer layer, the anionic polymer being selected from heparin, poly(sodium 4-styrenesulfonate) (PSS), or a mixture thereof, the cationic polymer being selected from chitosan or an analog thereof, and poly(allylamine hydrochloride) (PAH), or a mixture thereof, and the chitosan analog being N-alkylated chitosan; - functionalizing the outer layer pair of the multilayer polymer layer-by-layer (LbL) coating with heparin by terminal functionalization.

1. A method for preparing a blood compatible material, comprising:

21. An implantable device comprising a material according to any one of claims 1 to 19.

22. 22. The implantable device of claim 21, wherein the device is selected from stents, stent-grafts, endografts, vascular grafts, embolic filters, artificial blood vessels, drug delivery devices / balloons, patches, endovascular occlusion devices, CNS shunts, ventriculoperitoneal shunts, ventriculoatrial shunts, portosystemic shunts and shunts for ascites, heart valves and leaflets, shunts for pediatric cardiac surgery, vascular grafts and shunts for access surgery for dialysis, artificial hearts, and LVADs.

23. 20. An extracorporeal circulation device comprising the material of any one of claims 1 to 19.

24. - providing an implantable device or a portion thereof comprising a biocompatible nonwoven fabric as a support; - coating the substrate with a heparinized multi-polymer layer-by-layer (LbL) coating of the biocompatible nonwoven fiber, the multi-polymer coating comprising at least one layer pair of an anionic polymer layer and a cationic polymer layer, the multi-polymer layer-by-layer (LbL) coating being functionalized with heparin on an outer layer pair of the multi-polymer coating by terminal functionalization, the anionic polymer being selected from heparin, and poly(sodium 4-styrenesulfonate) (PSS), or a mixture thereof, the cationic polymer being selected from chitosan or an analog thereof, and poly(allylamine hydrochloride) (PAH), or a mixture thereof, and the chitosan analog being N-alkylated chitosan.

10. A method for preparing an implantable device, comprising:

25. 20. Use of a material according to any one of claims 1 to 19 for the preparation of an implantable device or an extracorporeal circulation device that comes into contact with blood.

26. Use of the method according to claim 20 or 24 for the preparation of an implantable device or an extracorporeal circulation device that comes into contact with blood.

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