Gradient coatings of biopeptides that promote endothelial cells selective adhesion and directional migration and methods of using the same

Abstract

A two-layer gradient coating article is provided that is operable to cause selective adhesion and directional migration of endothelial cells. The first layer includes cell-resisting polymers that repels cells, the second layer includes one layer of peptides that has affinity to and binds specifically to endothelial cells. Furthermore, the peptides are distributed in a gradient, in which attached ECs migrate towards the direction of increased concentration, thus enriching the ECs to a desired locus. The combination of a cell-repelling layer and a graded affinity peptide produces a unique result of selective adhesion, directional migration, thus local enrichment of endothelial cells. A method for using such gradient coating article and its potential use in treating cardiovascular diseases are also provided. The invention provides an inexpensive, stable and effective means for attracting ECs to desirable locations.

Claims

1. A gradient coating article configured to induce selective adhesion and directional migration of endothelial cells, comprising: a first layer of cell-resisting polymers that repels cell adhesion nondiscriminatory; and a second layer of peptides having specific affinity to, and operable to interact specifically with, endothelial cells, wherein the peptides are distributed in a concentration gradient configured to induce the endothelial cells to migrate towards the direction of increased peptide concentration at a rate that is higher than smooth muscle cells, and wherein the second layer is covalently linked to the first layer; wherein the first layer of cell-resisting polymers comprises evenly distributed polymer brushes of single polymer molecular thickness; and wherein the peptides are selected from the group consisting of Cys-Cys-Ala-Gly (SEQ ID NO: 3), Cys-Cys-Asn-Gly (SEQ ID NO: 4), Cys-Cys-Thr-Gly (SEQ ID NO: 5), Cys-Gly-Tyr-Leu (SEQ ID NO: 2), Cys-Ser-Val-Val-Tyr-Gly-Leu-Arg (SEQ ID NO: 6) and Cys-Arg-Glu-Asp-Val (SEQ ID NO: 1).

2. The gradient coating article according to claim 1, wherein the gradient coating is operable to repel at least 80% of non-endothelial cells and induce the adhesion of substantially all endothelial cells.

3. The gradient coating article according to claim 1, wherein the coating is operable to induce the directional migration of over 80% of endothelial cells, while exerting no impact on random migration of non-endothelial cells.

4. The gradient coating article according to claim 1, wherein the gradient coating is operable to induce the directional migration of the endothelial cells at a rate higher than when there is no gradient.

5. The gradient coating article according to claim 1, wherein the second layer of peptides is a single molecular layer.

6. The gradient coating article according to claim 1, wherein the first layer of cell-resisting polymers is linked to the second layer of peptides via conjugation by Michael addition reaction.

7. The gradient coating article according to claim 6, wherein the Michael addition reaction is between thiol group and carbon double bond.

8. The gradient coating article according to claim 1, wherein the cell-resisting polymers are selected from the following group: methacrylic anhydride modified hyaluronic acid with the molecular weight in the range of 500-200000 Da, and polyphosphoric acid choline with the molecular weight in the range of 500-200000 Da.

9. The gradient coating article according to claim 1, wherein the cell-resisting polymers have one end functionalized with a carboxyl group and have double bonds within repeating units.

10. The gradient coating article according to claim 1, wherein the thickness of the first layer of cell-resisting polymers is in the range of 0.5-5 nm.

11. The gradient coating article according to claim 1, wherein the thickness of the second layer of peptides is in the range of 0.5-5 nm.

12. The gradient coating article according to claim 1, further comprising: a substrate comprising biocompatible biodegradable polymers.

13. An implantable article configured to promote selective adhesion of endothelial cells and directional migration of the endothelial cells, comprising: a first layer of cell-resisting polymers that is uniform in composition and repels cell adhesion nondiscriminatory; a second layer of peptides having specific affinity to, and operable to interact specifically with, endothelial cells, wherein the peptides are distributed in a concentration gradient configured to induce the endothelial cells to migrate towards the direction of increased peptide concentration; and a substrate layer comprising biocompatible biodegradable polymers; wherein the peptides are selected from the group consisting of Cys-Cys-Ala-Gly (SEQ ID NO: 3), Cys-Cys-Asn-Gly (SEQ ID NO: 4), Cys-Cys-Thr-Gly (SEQ ID NO: 5), Cys-Gly-Tyr-Leu (SEQ ID NO: 2), Cys-Ser-Val-Val-Tyr-Gly-Leu-Arg (SEQ ID NO: 6) and Cys-Arg-Glu-Asp-Val (SEQ ID NO: 1).

14. The implantable article according to claim 13, wherein the endothelial cells migrate towards the direction of increased peptide concentration at a speed greater than that of smooth muscle cells.

15. The implantable article according to claim 13, wherein the first layer and the second layer are covalently linked.

16. The implantable article according to claim 13, wherein the implantable article is operable to form a part of an artificial blood vessel or stent to accelerate endothelium regeneration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 is schematic illustration showing the two layer structure of the gradient coating on a degradable biomaterial substrate, in accordance with certain embodiments.

(3) FIG. 2 is a schematic illustration showing the cell-repulsion effect of a cell-resisting polymer layer as well as selective adhesion and directional migration of ECs induced by the gradient coating, in accordance with certain embodiments.

(4) FIG. 3 is a graph showing .sup.1H NMR results of hyaluronic acid and methacrylate modified hyaluronic acid in the cell-resisting layer, in accordance with certain embodiments.

(5) FIG. 4 is a graph showing peptide density on uniform CREDV (Cys-Arg-Glu-Asp-Val) (SEQ ID NO: 1) surface as a function of thiol-ene reaction time, in accordance with certain embodiments.

(6) FIG. 5 is a graph showing molar ratio between CREDV (SEQ ID NO: 1) peptide to the repeating unit of MA-HA at different positions on the gradient surface, in accordance with certain embodiments. The result provides direct evidence of the successful generation of peptide concentration gradient.

(7) FIG. 6 is a graph showing representative fluorescent images of ECs (green) and smooth muscle cells (red) on CREDV (SEQ ID NO: 1) peptide concentration gradient in accordance with certain embodiments.

(8) FIG. 7 is a graph showing numbers of ECs and smooth muscle cells cultured for 8 hours on CREDV (SEQ ID NO: 1) concentration gradient, in accordance with certain embodiments. The quantitative result indicated that much more ECs adhered on the gradient coating compare to SMCs, proving the selective adhesion of ECs.

(9) FIG. 8 is a graph showing percentage of ECs or smooth muscle cells migrating towards +X direction on CREDV (SEQ ID NO: 1) concentration gradient, in accordance with certain embodiments. The result proved the selective directional migration of ECs was achieved by the gradient coating.

(10) FIG. 9 is a graph showing percentage of ECs or smooth muscle cells migrating towards +X direction on uniform CREDV (SEQ ID NO: 1) peptide surface with MA-HA, in accordance with certain embodiments. The result indicated that the uniform peptide surface cannot guide directional migration of ECs.

(11) FIG. 10 is a graph showing migration rate of ECs and smooth muscle cells on CREDV (SEQ ID NO: 1) concentration gradient, in accordance with certain embodiments. Compare with the result in FIG. 11, it clearly indicated that the migration rate of ECs was significantly enhanced by the gradient coating.

(12) FIG. 11 is a graph showing migration rate of ECs and smooth muscle cells on uniform CREDV (SEQ ID NO: 1) peptide surface with MA-HA, in accordance with certain embodiments.

(13) FIG. 12 is a graph showing numbers of ECs and smooth muscle cells cultured for 72 hours on CREDV (SEQ ID NO: 1) concentration gradient, in accordance with certain embodiments. Compared with FIG. 7, the results indicate a significant increase of ECs and nearly complete coverage of the surface (>80%) on the gradient coating as a result of combinational effect of selective adhesion and directional migration, while the number of SMCs was still very low.

(14) FIG. 13 is a graph showing numbers of ECs and smooth muscle cells cultured for 8 hours on MA-HA surface, in accordance with certain embodiments. The results suggested the universe cell-resisting effect to both ECs and SMCs of HA layer.

(15) FIG. 14 is a graph showing percentage of ECs or smooth muscle cells migrating towards +X direction on MA-HA surface, in accordance with certain embodiments. The result indicated that the cell-resisting layer does not have ability to induce directional migration of both ECs and SMCs.

(16) FIG. 15 is a graph showing migration rate of ECs and smooth muscle cells on MA-HA surface, in accordance with certain embodiments. The result indicated that SMCs move much faster than ECs on cell-resisting layer, which is similar to the pattern in natural environment.

(17) FIG. 16 is a graph showing numbers of ECs and smooth muscle cells cultured for 72 hours on MA-HA surface, in accordance with certain embodiments. Together with FIG. 13, the results suggested the long term cell-resisting effect to both ECs and SMCs of HA layer.

(18) FIG. 17 is a graph showing numbers of ECs and smooth muscle cells cultured for 8 hours on CREDV (SEQ ID NO: 1) peptide concentration gradient without MA-HA, in accordance with certain embodiments. The result indicated that the peptide concentration gradient can support both ECs and SMCs adhesion without the HA cell-resisting layer.

(19) FIG. 18 is a graph showing percentage of ECs or smooth muscle cells migrating towards +X direction on CREDV (SEQ ID NO: 1) peptide density gradient without MA-HA surface, in accordance with certain embodiments. The result indicated that peptide concentration gradient alone can selectively induce directional migration of ECs to some extent, but the effect is smaller than gradient coating.

(20) FIG. 19 is a graph showing migration rate of ECs and smooth muscle cells on CREDV (SEQ ID NO: 1) peptide concentration gradient without MA-HA, in accordance with certain embodiments. The result indicated that peptide concentration gradient alone cannot enhance the migration rate of ECs and SMCs.

(21) FIG. 20 is a graph showing numbers of ECs and smooth muscle cells cultured for 72 hours on CREDV (SEQ ID NO: 1) peptide concentration gradient without MA-HA, in accordance with certain embodiments. The result indicated that peptide concentration gradient alone can enhance the number of both ECs and SMCs, without cell selectivity.

(22) FIG. 21 is a graph showing numbers of ECs and smooth muscle cells cultured for 8 hours on CREDV (SEQ ID NO: 1)/HA complimentary gradient, in accordance with certain embodiments. The result indicated that the complimentary gradient can partially reject both ECs and SMCs adhesion to similar extent. But this effect is very low compare to that of the gradient coating (FIG. 7). The cell selectivity is also poor. The limited effect is mainly attributed to the low concentration of HA cell-resisting molecules on the most part of the gradient.

(23) FIG. 22 is a graph showing numbers of ECs and fibroblasts cultured for 8 hours on CREDV (SEQ ID NO: 1) concentration gradient, in accordance with certain embodiments. The result indicated that more ECs adhered on the gradient coating compare to FIBs, proving again the selective adhesion of ECs.

(24) FIG. 23 is a graph showing percentage of ECs or fibroblasts migrating towards +X direction on concentration density gradient of CREDV (SEQ ID NO:1) in accordance with certain embodiments. The result proved again the selective directional migration of ECs was achieved by the gradient coating.

(25) FIG. 24 is a graph showing migration rate of ECs and fibroblasts on CREDV (SEQ ID NO: 1) concentration gradient in accordance with certain embodiments. Compared to FIG. 11, the result clearly indicated that the migration rate of ECs was selectively enhanced by the gradient coating.

(26) FIG. 25 is a graph showing numbers of ECs and fibroblasts cultured for 72 hours on CREDV (SEQ ID NO: 1) concentration gradient in accordance with certain embodiments.

(27) FIG. 26 is a graph showing characterization of polyurethane artificial vascular grafts and images of artificial vascular grafts sutured onto rat abdominal aorta.

(28) FIG. 27 is a graph showing H&E staining of artificial vascular grafts modified with (a) MA-HA, (b) CREDV (SEQ ID NO: 1) peptide concentration gradient without MA-HA layer, and (c) CREDV (SEQ ID NO: 1) peptide concentration gradient with MA-HA layer after being planted in vivo for 1 week, in accordance with certain embodiments. The result indicated that fully regeneration of endothelium is achieved on artificial vessel via concomitant MA-HA and CREDV (SEQ ID NO: 1) peptide gradient.

DETAILED DESCRIPTION OF THE INVENTION

(29) It is to be understood that certain descriptions of the present invention have been simplified to illustrate only those elements and limitations that are relevant to a clear understanding of the present invention, while eliminating, for purposes of clarity, other elements. Those of ordinary skill in the art, upon considering the present description of the invention, will recognize that other elements and/or limitations may be desirable in order to implement the present invention. However, because such other elements and/or limitations may be readily ascertained by one of ordinary skill upon considering the present description of the invention, and are not necessary for a complete understanding of the present invention, a discussion of such elements and limitations is not provided herein. As such, it is to be understood that the description set forth herein is merely exemplary to the present invention and is not intended to limit the scope of the claims.

(30) Other than in the examples herein, or unless otherwise expressly specified, all of the numerical ranges, amounts, values, and percentages, such as those for amounts of materials, elemental contents, times and temperatures of reaction, ratios of amounts, and others, in the following portion of the specification and attached claims may be read as if prefaced by the word about, even though the term about may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains error necessarily resulting from the deviation found in its underlying respective testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (i.e., end points may be used). Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of 1 to 10 is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

(31) Certain compositions within the present invention are generally described in the form of biopolymers for use in medical and biological systems. It will be understood, however, that the present invention may be embodied in forms and applied to end uses that are not specifically and expressly described herein. For example, one skilled in the art will appreciate that compositions and methods comprising plastics have application in many industries, as well as the medical arts.

(32) All patents, publications, or other disclosure material referenced herein are incorporated by reference in their entirety. Any patent, publication, or other disclosure material, in whole or in part, that is incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.

(33) The articles a, an, and the are used herein to refer to one or more than one (i.e., to at least one) of the grammatical objects of the article. By way of example, a component means one or more components, and thus, possibly, more than one component is contemplated and may be employed or used.

(34) The term polypeptide, and the terms protein and peptide which are used interchangeably herein, refers to a polymer of amino acids.

(35) The term polymer, as used herein, refers to natural and synthetic molecules with repeating structural units including, but not limited to, molecules comprising gels and plastics.

(36) The term biocompatible refers to the absence of stimulation of a severe, long-lived or escalating biological response to an implant or coating, and is distinguished from a mild, transient inflammation which typically accompanies surgery or implantation of foreign objects into a living organism.

(37) The term degradable biomaterial refers to biocompatible materials that are degradable within a live organism including a human being. Examples of degradable biomaterial include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolytic acid (PLGA), polycaprolactone, and copolymers thereof, polyesters, such as polyglycolides, polyanhydrides, polyacrylates, polyalkyl cyanoacrylates, such as n-butyl cyanoacrylate and isopropyl cyanoacrylate, polyacrylamides, polyorthoesters, polyphosphazenes, polypeptides, polyurethanes, polystyrenes, polystyrene sulfonic acid, polystyrene carboxylic acid, polyalkylene oxides, alginates, agaroses, dextrins, dextrans, and polyanhydrides.

(38) A gradient is one or more dimensions in space along which the concentration and/or accessibility of one or more substances may vary. Concentration gradients include both linear gradients (i.e., gradients which increase or decrease at a continuous rate) and non-linear gradients.

(39) The present invention provides a gradient coating which, when applied a medium or standing alone, can cause selective adhesion and directional migration of ECs, which can be used to support the regeneration of the endothelium on artificial vessel or implant stent. The medium includes the surfaces of degradable biomaterial, a film, a medical device, or a tissue, or a layer of cells.

(40) The gradient coating comprises a two-layer structure, in which the lower layer is consist of certain specific cell-resisting polymers and the upper layer is consist of density gradient of peptides which is able to selectively interact with ECs. The gradient coating can induce the selective adhesion of ECs and subsequently direction migration toward +X direction of the gradient, said +X direction of the gradient is the direction with enhanced peptide density.

(41) In some embodiments, a uniformly distributed single molecular thick cell-resisting polymer layer is provided to repel cells, i.e., to provide an anti-cell adhesion function that prevent adhesion of non-targeted cells such as SMCs. Preferably, the cell-resisting polymer is selected from following polymers: methacrylicanhydride modified hyaluronic acid (MA-HA) and polyphosphoric acid choline, with the molecular weight in the range of 500-200000 Da. These polymers can be synthetic, derived from natural extracellular matrix, or both. One example of the cell-resisting polymer is hyaluronic acid (HA), a natural polysaccharide that widely used in medical applications due to its superior inert properties as an anionic glycosaminoglycan and high antifouling ability against fibronectin adsorption. Therefore, the MA-HA layer functions to resist non-selective cell adhesion. In some embodiments, only one single molecular layer with the thickness within 0.5-5 nm of the cell-resisting polymer is enough to achieve satisfactory cell-resist effect. The cell-resisting polymers contain carboxyl group and double bond in their repeating unit, feasible for the reaction with amino group functionalized substrate and thiol group functionalized peptide, respectively.

(42) In some embodiments, thiol (cysteine)-functionalized peptide which can specifically interact with ECs was conjugated onto the cell-resisting polymer layer containing double bonds via a Michael addition reaction, a high efficiency reaction, in a gradient manner to form the density gradient of peptides. In some embodiments, the peptides are selected from following sequences: Cys-Cys-Ala-Gly (SEQ ID NO: 3), Cys-Cys-Asn-Gly (SEQ ID NO: 4), Cys-Cys-Thr-Gly (SEQ ID NO: 5), Cys-Gly-Tyr-Leu (SEQ ID NO: 2), Cys-Ser-Val-Val-Tyr-Gly-Leu-Arg (SEQ ID NO: 6), Cys-Arg-Glu-Asp-Val (SEQ ID NO: 1). In a preferred embodiment, a Cys-Arg-Glu-Asp-Val (CREDV) (SEQ ID NO: 1) peptide is used, which is the smallest active sequence of fibronectin that is recognized by and interacts with integrin .sub.4.sub.1 receptor on ECs and has been widely used for designing EC-selective surfaces. In come embodiments, a single layer peptide with the thickness in the range of 0.5-5 nm is provided. These peptides are synthetic, very short sequence with low cost, relatively easy to manufacture and stable in their application, compared to expensive and unstable proteins/growth factors. In addition, the small size of the peptide allows refined tunable binding density on the cell-resisting polymer layer, leading to excellent control of the density gradient of the peptide.

(43) With the presence of cell-resisting polymer layer, over 80% of cells are repelled or rejected from the polymer surface, preventing them from strong adhesion. The said gradient coating can support the adhesion of ECs, but cannot support the adhesion of other cells such as SMCs and fibroblasts. Besides, the gradient coating also can induce the directional migration of ECs, with over 80% of ECs continuously move toward the direction with enhanced peptide density. Meanwhile the gradient coating has no influence on the random movements of other cells. The gradient coating also enhances the migration rate of ECs, as a result the ECs have a higher migration rate gradient coating on the compared to that of SMCs.

(44) In some embodiments, a gradient material, which can induce selective adhesion and directional migration of ECs, was generated by covalently immobilizing said gradient coating onto a degradable biomaterial with amino groups on its surface, in the presence of a proper crosslinking agent. The obtained gradient material can induce the selective adhesion of ECs and subsequently direction migration toward +X direction of the gradient, with enhanced peptide density.

(45) An implantable material, which contains the above mentioned gradient material, can be generated and used for artificial blood vessel or stent.

(46) A method to prepare the gradient materials which can selectively control the adhesion and directional migration of ECs, said method includes following steps: 1) Prepare cell-resisting polymer solution and covalently conjugate the cell-resisting polymer onto substrate, said substrate is biocompatible biodegradable polymers. 2) Prepare peptide solution and gradually conjugate onto the cell-resisting polymer layer with covalent bonding to form a concentration gradient of peptide, said peptide is able to selectively interact with ECs. As a result, said gradient material which can induce selective adhesion of ECs and subsequently direction migration toward +X direction of the gradient, said +X direction of the gradient is the direction with enhanced peptide density.

(47) The following examples are intended to more clearly illustrate aspects of the invention, but are not intended to limit the scope thereof.

Example I

Synthesis of Methacrylatedhyaluronic Acid (MA-HA)

(48) MA-HA was prepared according to a previously reported method. [Bae, M.; Yang, D.; Lee, J.; Heo, D.; Kwon, Y.; Youn, I. Photo-cured Hyaluronic Acid-Based Hydrogels Containing Simvastatin as a Bone Tissue Regeneration Scaffold. Biomaterials 2011, 32, 8161-8171.] In brief, HA (1.0 g) was dissolved in 100 mL mixed solvent of DMF/water (1/2, v/v), into which a 20-fold excess of methacrylic anhydride (7.4 mL) was added. The mixture was stirred for 24 h in an ice bath after the solution pH was adjusted to 8.0 using a 5 M NaOH solution. The pH was maintained between 8-9 during the whole procedure. MA-HA was obtained by precipitation in excess ethanol and collected by centrifugation, which was further purified by dialysis for 3 d against distilled water, and finally freeze-dried. The grafting ratio of methacrylate was 2.1 in each HA repeating unit with four OH reaction sites according to .sup.1H NMR (Figure. 3).

Preparation of Polydopamine (PDA) Coated PCL Substrate

(49) PCL membrane was prepared by spin-coating a 2% (w/v) PCL/1,4-dioxane solution onto the glass slides under 1600 rpm. The PCL membranes were immersed in a 2 mg/mL dopamine/tris-HCl (10 mM, pH=8.5) solution at 37 C. After a 12 h deposition, the PDA-coated PCL membranes were washed with water.

Grafting of MA-HA

(50) MA-HA molecules were immobilized onto the surface of the PCL-PDA substrate via a condensation reaction between COOH of MA-HA and NH.sub.2 of PDA under the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS). In brief, 200 mg MA-HA, 7 mg EDC and 12 mg NHS were dissolved in 100 mL water, and its pH value was adjusted to 5.5, in which the PDA-coated PCL membranes were incubated at 37 C. for 3 h, followed by washing with plenty of water.

Grafting of CREDV Peptides

(51) Thiol-functionalized CREDV (SEQ ID NO: 1) peptides were dissolved in Na.sub.2CO.sub.3/NaHCO.sub.3 buffer (pH=9) to reach a concentration of 0.4 mg/mL. The MA-HA-coated PCL membranes were immersed into the CREDV (SEQ ID NO: 1) solution at 37 C. for different times. To generate an CREDV (SEQ ID NO: 1) concentration gradient (total length was 10 mm), the CREDV (SEQ ID NO: 1) solution was slowly injected into a centrifuge tube by a micro-infusion pump (WZS-50F2 from Zhejiang University Medical Instrument, Hangzhou, China) at 37 C., where MA-HA-coated PCL membranes were placed vertically. The injection rate was adjustable with the longest reaction time of 60 min.

Cell Adhesion Assay

(52) The co-culture of ECs and smooth muscle cells was implemented by seeding 2.110.sup.4/cm.sup.2 of each type of cell in a 1:1 mixture of RPMI-1640 and DMEM. ECs and Smooth muscle cells were stained with 5 mg/mL 3,3-dioctadecyloxacarbocyanine perchlorate (DiO, green) and 1,1-dioctadecyl-3,3,3,3-tetramethyl indocarbocyanine perchlorate (DiL, red), respectively, for 5 min before seeding.

(53) As shown in FIGS. 6 and 7, the number of ECs was approximately 2.5 folds of that of smooth muscle cells on the whole CREDV (SEQ ID NO: 1) concentration gradient, indicating the selective cell adhesion effect achieved by gradient coating.

Cell Migration Assay

(54) The respective uniform surfaces with the same reaction time and thereby the same CREDV (SEQ ID NO: 1) peptide concentrations were used as controls. ECs and smooth muscle cells were seeded at a density of 510.sup.3/cm.sup.2 and allowed to attach for 12 h. Cell migration traces were recorded in situ for 12 h under a time-lapse phase-contrast microscope equipped with a cell culture chamber (37 C. and 5% CO.sub.2 humidified atmosphere). The starting position of each single cell was automatically defined as the original position (0, 0). The cell migration distance S was then calculated by the Chemotaxis Tool (Ibids, Germany) according to the following equation.
S=.sub.i=1.sup.t{square root over ((x.sub.ix.sub.i-1).sup.2+(y.sub.iy.sub.i-1).sup.2)}(1)

(55) The cell migration rate was calculated base on the formula v=S/t. [Han, L.; Mao, Z.; Wu, J.; Guo, Y; Ren, T.; Gao, C. Directional Cell Migration through Cell-cell Interaction on Polyelectrolyte Multilayers with Swelling Gradients. Biomaterials 2013, 34, 975-984.]

(56) As shown in FIGS. 8 and 9, 84% of the ECs moved toward the higher CREDV (SEQ ID NO: 1) peptide concentration of the gradient, which is much higher than smooth muscle cells. On uniform surface, both around 50% ECs and smooth muscle cells migrated towards +X direction. The results proved the selective directional migration of ECs was achieved by the gradient coating.

(57) As shown in FIGS. 10 and 11, the migration rate of smooth muscle cells slowed down slightly (11.8 m/h). On CREDV (SEQ ID NO: 1) peptide concentration gradient, endothelial cells migrated faster than smooth muscle cells. The results clearly indicated that the migration rate of ECs was significantly enhanced by the gradient coating.

(58) As shown in FIG. 12, after being cultured for 72 h, number of ECs was almost 5-times to smooth muscle cells on CREDV (SEQ ID NO: 1) concentration gradient. The results indicate the continuous increase of ECs on the gradient coating as a result of combinational effect of selective adhesion and directional migration, while the number of SMCs was still very low.

Comparing Example I

(59) Grafting of MA-HA was carried out according to Example 1. No CREDV (SEQ ID NO: 1) peptide was further immobilized.

(60) Cell adhesion and migration assays were carried out according to Example 1.

(61) As shown in FIG. 13, the cell adhesion number of ECs and smooth muscle cells was similar on MA-HA surface, which was much lower than ECs on CREDV (SEQ ID NO: 1) concentration gradient. The results suggested the universe cell-resisting effect to both ECs and SMCs of HA layer.

(62) As shown in FIG. 14, both around 50% of ECs and smooth muscle cells migrated towards +X direction. ECs showed no directional migration on MA-HA surface. The result indicated that the cell-resisting layer do not have ability to induce directional migrating of both ECs and SMCs.

(63) As shown in FIG. 15, migration rate of ECs (7 m/h) was much lower compared to CREDV (SEQ ID NO: 1) concentration gradient (14 m/h). The result also indicated that SMCs move much faster than ECs on cell-resisting layer, which is similar to the pattern in natural environment.

(64) As shown in FIG. 16, after being cultured for 72 h, the number of ECs and smooth muscle cells on MA-HA surface was similar, which was only of the number of ECs on CREDV (SEQ ID NO: 1) concentration gradient. Together with FIG. 13, the results suggested the long termcell-resisting effect to both ECs and SMCs of HA layer.

Comparing Example II

(65) To generate an CREDV (SEQ ID NO: 1) concentration gradient (total length was 10 mm) without cell-resisting layer, the CREDV (SEQ ID NO: 1) solution was slowly injected into a centrifuge tube by a micro-infusion pump at 37 C., where PDA-coated PCL membranes were placed vertically, The injection rate was adjustable with the longest reaction time of 60 min. As a result, a CREDV (SEQ ID NO: 1) concentration gradient without MA-HA, the cell-resisting layer, was fabricated.

(66) Cell adhesion and migration assays were carried out according to Example 1.

(67) As shown in FIG. 17, on CREDV (SEQ ID NO: 1) peptide concentration gradient without MA-HA, the number of smooth muscle cells was much higher than that on CREDV (SEQ ID NO: 1) concentration gradient. The result indicated that the peptide concentration gradient can support both ECs and SMCs adhesion without the HA cell-resisting layer.

(68) As shown in FIG. 18, on CREDV (SEQ ID NO: 1) peptide concentration gradient without MA-HA, 70% of ECs migrated towards +X direction, which was much lower than on CREDV (SEQ ID NO: 1) concentration gradient with MA-HA.

(69) As shown in FIG. 19, migration rate of ECs on CREDV (SEQ ID NO: 1) concentration gradient without MA-HA was much lower than smooth muscle cells. The result indicated that peptide concentration gradient alone can selectively induce directional migration of ECs to some extent, but the effect is smaller than gradient coating (FIG. 8).

(70) As shown in FIG. 20, after being cultured for 72 h, the number of ECs was similar to that of smooth muscle cells, which showed no cell selectivity.

Comparing Example III

(71) A complimentary gradient coating with HA and REDV (SEQ ID NO: 1) peptide was generated and compared, in according to [Ren, T.; Yu, S.; Mao, Z.; Enrique Moya, S.; Han, L.; Gao, C. Complementary Density Gradient of Poly(hydroxyethyl methacrylate) and YIGSR Selectively Guides Migration of Endotheliocytes. Biomacromolecules 2014, 15, 2256-2264.]

(72) A PDA coated PCL membrane, prepared according to example I, was vertically placed in a cylindrical plastic container, into which the HA solution with the presence of EDC/NHS was injected continuously by a micro infusion pump at room temperature with a rate of 2 mL/h. A total of 3 h later, the membrane with HA concentration gradient was taken out and washed with plenty of water. The membrane was then immersed in a glutaraldehyde solution to react with residual amino groups and change them to aldehyde group. After thoroughly washing in plenty of water, the membrane was immersed into a CREDV (SEQ ID NO: 1) solution to generate a complimentary concentration gradient of peptide.

(73) Cell adhesion and migration assays were carried out according to Example 1.

(74) As shown in FIG. 21, the results showed that the complimentary gradient can partially reject both ECs and SMCs adhesion to similar extent. But this effect is very low compare to that of the gradient coating (FIG. 7). Besides, the cell selectivity is also very poor. The limited effect is mainly attributed to the low concentration of HA cell-resisting molecules on the most part of the gradient.

Comparing Example IV

(75) The grafting of MA-HA and CREDV (SEQ ID NO: 1) peptide were carried out according to Example 1. The CREDV (SEQ ID NO: 1) peptide concentration gradient was fabricated according to Example 1.

Cell Adhesion Assay

(76) The co-culture of ECs and fibroblasts was implemented by seeding 2.110.sup.4/cm.sup.2 of each type of cell in a 1:1 mixture of RPMI-1640 and DMEM. ECs and Smooth muscle cells were stained with 5 mg/mL DiO (green) and DiL (red), respectively, for 5 min before seeding.

(77) As shown in FIG. 22, the number of ECs was approximately 2 folds of that of fibroblasts on the whole CREDV (SEQ ID NO: 1) density gradient, confirmed again the cell selective adhesion induced by the gradient coating.

(78) Cell migration assay were carried out according to Example 1.

(79) As shown in FIG. 23, 84% of the ECs moved toward the higher CREDV (SEQ ID NO: 1) peptide concentration of the gradient, which is much higher than fibroblasts. The result proved again the selective directional migration of ECs was achieved by the gradient coating.

(80) As shown in FIG. 24, endothelial cells migrated faster than smooth muscle cells on CREDV (SEQ ID NO: 1) peptide concentration gradient. Compared to FIG. 11, the result clearly indicated that the migration rate of ECs was selectively enhanced by the gradient coating.

(81) As shown in FIG. 25, after being cultured for 72 h, number of ECs was almost 4-times to fibroblasts on CREDV (SEQ ID NO: 1) peptide concentration gradient.

Example 2

Grafting of Polyphosphoric Acid Choline

(82) Polyphosphoric acid choline molecules were immobilized onto the surface of the PDA-coated PCL membrane via a condensation reaction between COOH of polyphosphoric acid choline and NH.sub.2 of PDA under the presence of EDC/NHS. In brief, 200 mg polyphosphoric acid choline, 7 mg EDC and 12 mg NHS were dissolved in 100 mL water, and its pH value was adjusted to 5.5, in which the PDA-coated PCL membranes were incubated at 37 C. for 3 h, followed by washing with plenty of water.

Grafting of CGYL Peptides

(83) To generate an CGYL (Cys-Gly-Tyr-Leu) (SEQ ID NO: 2) concentration gradient (total length was 10 mm), the CGYL (SEQ ID NO: 2) solution was slowly injected into a centrifuge tube by a micro-infusion pump at 37 C., where polyphosphoric acid choline-coated PCL membranes were placed vertically. The injection rate was adjustable with the longest reaction time of 60 min.

(84) Cell adhesion and migration assays were carried out according to Example 1.

(85) The number of ECs was approximately 2.3 fold of that of smooth muscle cells on the whole CGYL (SEQ ID NO: 2) concentration gradient.

(86) 86% of the ECs moved toward the higher CGYL (SEQ ID NO: 2) peptide concentration of the gradient, which is much higher than the smooth muscle cells (46%).

(87) The migration rate of the smooth muscle cells slowed down slightly (10.4 m/h). On CGYL (SEQ ID NO: 2) peptide concentration gradient, ECs (13.7 m/h) migrated faster than the smooth muscle cells.

(88) After being cultured for 72 h, number of ECs was almost 4.2-times higher than smooth muscle cells on CGYL (SEQ ID NO: 2) concentration gradient.

Example 3

Preparation of PDA-Coated Polyurethane (PU) Artificial Vascular Graft

(89) PU tubes were prepared by dip-coating a 2% (w/v) PU/hexafluoroisopropanol solution mixed with 4% (w/v) NaCl onto the stainless steel cylinders with a diameter of 1.8 mm. The obtained PU tubes were immersed in a 2 mg/mL dopamine/tris-HCl (10 mM, pH=8.5) solution at 37 C. After a 12 h deposition, the PDA-coated PU tubes were removed and washed with water.

(90) Grafting of MA-HA cell-resisting layer and CREDV (SEQ ID NO: 1) concentration gradient on PDA coated PU vessel were carried out according to Example 1.

(91) As shown in FIG. 26, rat abdominal aorta was cut into two parts, and the artificial vascular grafts were sutured onto the two ends of aorta. After 1 week, the artificial grafts were taken out and characterized with H&E staining

(92) As shown in FIG. 27, the artificial vascular grafts coating with CREDV (SEQ ID NO: 1) concentration gradient showed no thrombus compared to the ones coating with MA-HA. An intact endothelium was regenerated on the gradient coating. In contrast, an obvious thrombosis was observed in the vessels with only cell-resisting coating or with only peptide concentration gradient. No regeneration of endothelium was observed. The results showed the gradient material including MA-HA and CREDV (SEQ ID NO: 2) peptide concentration gradient can synergistically help the regeneration of endothelium, and may be used in artificial vascular grafts modification.

(93) While the invention has been particularly shown and described as referenced to the embodiments thereof, those skilled in the art will understand that the foregoing and other changes in form and detail may be made therein without departing from the spirit and scope.