Patent application title: FUNCTIONAL NANOSTRUCTURED CHITOSAN COATINGS FOR MEDICAL INSTRUMENTS AND DEVICES
Nano And Advanced Materials Institute Limited (Hong Kong, HK)
Ka Wai Wong (Hong Kong, HK)
Mui Kimmy Chan (Hong Kong, HK)
Ching Yee Novem Lam (Hong Kong, HK)
Nano and Advanced Materials Institute Limited
IPC8 Class: AC25D902FI
Class name: Drug, bio-affecting and body treating compositions preparations characterized by special physical form
Publication date: 2013-08-08
Patent application number: 20130202660
Disclosed herein is an approach for coating a biocompatible material, in
particular chitosan, onto a surface in need thereof. This approach
results in a coating that is highly uniform, even on irregular surfaces,
and enables ready adjustment of the morphology of the coated material. A
convenient means to add a functional property to the biocompatible
material by incorporation of functional additives is also provided.
1. A method for coating a biopolymer in an adjustable morphology onto a
metal-based surface in need thereof comprising: providing an electrolyte,
said electrolyte comprising the biopolymer which is dissolved in a
non-aqueous solvent system containing a supporting electrolyte and at
least one biopolymer functional additive; providing a negative counter
electrode, a reference electrode and a positive working electrode;
contacting said electrolyte with the counter electrode, reference
electrode, and working electrode; and applying an electrical potential
and current, such that the biopolymer deposits onto the surface of said
positive working electrode in order to form a coating of said biopolymer
and the functional additive in an adjustable morphology.
2. The method of claim 1, wherein said biopolymer is chitosan.
3. The method of claim 2 further comprises adjusting a pH of said electrolyte to a pH of less than 6.5.
4. The method of claim 3, wherein the pH of said electrolyte is adjusted to pH 4 to 4.5.
5. The method of claim 1, wherein said non-aqueous solvent system is propylene carbonate: ethylene glycol in a ratio of 1:4 v/v.
6. The method of claim 1, wherein the supporting electrolyte comprises LiClO4, KCL, KNO3, HCl, NaOH, tetraalkylammonium salts or a combination thereof.
7. The method of claim 6, wherein a concentration of the supporting electrolyte is 5.5 mM to 55 mM.
8. The method of claim 1, wherein said surface of the positive working electrode is a conductive surface comprising pure metals or metal alloys.
9. The method of claim 8, wherein said surface of the positive working electrode is stainless steel-based or titanium-based.
10. The method of claim 2, wherein the morphology of chitosan coating includes one or more of particles, wires, rods, flakes, ribbons, flower-like, urchin-like, multipods or tubes.
11. The method of claim 1, wherein said at least one functional additive is 5-florouracil.
12. The method of claim 1, wherein said at least one functional additive is calcium carbonate.
13. The method of claim 11, wherein the ratio of said biopolymer to said 5-florouracil is about 1:8.
14. The method of claim 12, wherein the ratio of said biopolymer to said calcium carbonate is about 1:5.
15. A medical device coated by the method of claim 1, wherein the medical device is configured for implantation or insertion into an animal in need thereof.
CROSS REFERENCE TO RELATED APPLICATIONS
 The present application claims priority of U.S. provisional application No. 61/633,141 filed on Feb. 6, 2012, the disclosure of which is herein incorporated by reference.
FIELD OF THE INVENTION
 The present invention relates to methods of coating biopolymer with at least one functional additive onto surfaces in need thereof and more particularly to surfaces of medical instruments and devices. The present invention also relates to medical devices coated by the present methods and uses thereof.
 Medical instruments and devices, such as medical implants, are very often coated with one or more coatings to impart biocompatibility, enable drug delivery, provide lubricity and so on. Various research groups focus on development of different nanostructure coatings for medical implants. For instance, Seoul National University discloses inorganic nanostructured coatings on titanium for preosteoblastic cell growth (Journal of Biomedical Materials Research, Part A, Vol. 90A, Issue 4, 2008, Pages 1239-1242). Nano-sized hydroxyapatite coatings on titanium substrates that facilitate osteoblast cell proliferation have also been reported (Journal of the American Ceramic Society, Vol. 90, Issue 1, 2007, Pages 50-56).
 Chitosan, a polycationic biopolymer of (1-4)-linked 2-amino-2deoxy-D-glucopyranose, is well-known for its versatile applications in biosensing, medicine and pharmaceuticals due to its superior bioavailability and biocompatible characteristics. There are many techniques for coating chitosan onto various surfaces of medical instruments. Feng et al. of Southeast University of China demonstrated the usefulness of electrospun chitosan nanofibers for hepatocyte cultures (Journal of Biomedical Nanotechnology, Vol. 6, No. 6, 2010, Pages 1-9). Kumbar et al. of University of Virginia successfully demonstrated positive results in engineering soft tissues using electrospun nanofiber scaffolds (Biomedical Materials, Vol. 3, 2008).
 However, many of the conventional biopolymer coating techniques, such as spincasting, electrospinning and spraying, result in coatings lacking uniformity, especially on irregular shapes which are very often present in medical instruments. Furthermore, the conventional coating techniques are very limited in terms of morphology or nanostructure of the resulting chitosan. Morphology of the resulting chitosan coating prepared by conventional methods is typically limited to a single form. That is, the morphology cannot be tuned and customized for the specific functions of the particular medical instruments coated therewith. Therefore, there is a need for a novel coating technique for biopolymers that overcomes the shortcomings of the existing techniques.
SUMMARY OF THE INVENTION
 One aspect of the present invention is to provide a novel method for coating biopolymers having at least one functional additive onto surfaces in need of such coatings. The biopolymer coating prepared according to the present method is highly uniform. In particular, the biopolymer coating prepared by the present method can take the form of a single crystal whose morphology or nanostructure can be adjusted in order to customize and facilitate the functions of surfaces being coated. The present method of coating a biopolymer having at least one functional additive onto an article in need thereof comprises providing an electrolyte in which the biopolymer is dissolved in a non-aqueous solvent system, and a supporting electrolyte, adjusting the electrolyte to an acidic pH, providing a negative counter electrode, a reference electrode and a positive working electrode, contacting the electrolyte solution with the negative counter electrode, reference electrode and positive working electrode and applying an electric field such that the biopolymer deposits onto the positive working electrode to form a coating. The positive working electrode is the article to be coated. The present method further comprises adding at least one functional additive to the electrolyte. The present method of coating enables the morphology of the biopolymer coating to be tuned and adjusted to facilitate the application of the article being coated. The present coating method also provides a convenient means to add functional properties to the biopolymer coating by incorporation of one or more different functional additives.
 Another aspect of the present invention is to provide articles coated with a biopolymer prepared by the present method of coating. The article is a medical instrument or device. In an exemplary embodiment, the article is an implantable or invasive medical device having a metallic surface or which is itself metal-based.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 shows an arrangement of the electrolyte and electrodes of the present coating method.
 FIG. 2 shows an SEM picture of a chitosan coating incorporated with 5-florouracil prepared by the present method of coating on a stainless steel surface. FIG. 2A and FIG. 2B are chitosan 5-florouracil crystal prepared with a 5.5 mM LiClO4 supporting electrolyte at magnifications of ×2,000 and ×14,000, respectively. FIG. 2C and FIG. 2D are chitosan 5-florouracil crystal prepared with a 55 mM LiClO4 supporting electrolyte at magnifications of ×1,500 and ×35,000, respectively.
 FIG. 3 depicts HeLa cell viability test results on a bare stainless steel article (a), a stainless steel article coated with sparse chitosan by the present method (at 5.5 mM LiClO4) (b), a stainless steel article coated with dense chitosan by the present method (at 55 mM LiClO4) (c), a stainless steel article coated with sparse chitosan and 5-florouracil by the present method (at 5.5 mM LiClO4) (d), and a stainless steel article coated with dense chitosan and 5-florouracil by the present method (at 55 mM LiClO4) (e). The time period is three days.
 FIG. 4A shows an SEM picture of a chitosan coating incorporating calcium carbonate prepared by the present method on stainless steel, magnification of ×15,000; FIG. 4B depicts MC3T3-E1 cell viability test results on titanium alone (Titanium), stainless steel coated with chitosan by the present method (without CaCO3) and stainless steel coated with chitosan and calcium carbonate by the present method (with CaCO3).
DETAILED DESCRIPTION OF THE INVENTION
 Reference will now be made in detail to the exemplary embodiments of the present invention, serve to explain the principles of the invention. These embodiments or examples are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized, and that changes may be made without departing from the spirit of the present invention.
 The present invention pertains to a method for coating biopolymers onto surfaces in need thereof. In one aspect, the method comprises providing an acidic electrolyte having the biopolymer to be coated dissolved in a non-aqueous solvent system and a supporting electrolyte, providing a negative counter electrode, a reference electrode and a positive working electrode, contacting the electrolyte with the negative counter electrode, reference electrode and positive working electrodes and applying appropriate electrical potential and current, wherein the biopolymer deposits onto the working electrode, forming a coat thereon. The positive working electrode is the article being coated. In one embodiment, ultrasonication may be employed to facilitate proper dissolution of chitosan in the solvent system.
 The biopolymer coatings prepared according to the present method are highly uniform, even when coated on irregularly shaped surfaces. In particular, the biopolymer coatings prepared by the present method can take the form of a single crystal, wherein the morphology or nanostructure of the single crystal of biopolymer can be adjusted in order to customize and facilitate the functions of the surfaces being coated. Various biopolymers can be coated onto substrates according to the methods of the present invention. Examples of biopolymers that can be coated by the present invention include any biocompatible compounds bearing at least one chemical functional groups for intra- and/or inter-hydrogen bond formation. Examples of the at least one chemical functional groups include, but are not limited to, --NH2, --COON and --OH. In one embodiment, the biopolymer is chitosan. The term "chitosan" as used herein, may refer to pure chitosan, a chitosan salt, a chitosan derivative or a combination thereof. Chitosan for use in the present invention may be obtained from natural sources, such as shells of shrimps and crabs by deacetylation of natural chitin in hydrogen peroxide solution or other techniques which are known in the art. Alternatively, chitosan may be made synthetically or may be chemically modified by other techniques known in the art. The chitosan coated by the present invention may be a mixture of different forms of chitosan or a homogeneous coating of one chitosan, so long as the chitosan(s) can be dissolved completely in a non-aqueous solvent system. In certain embodiments, chitosan suitable for use in the present invention may have a molecular weight in the range of from 5 kDa to 2000 kDa, or from 50 kDa to 1000 kDa or from 100 kDa to 900 kDa. In certain embodiments, the chitosan may have a percentage of deacetylation from 40% to 100%. In one embodiment, the chitosan has greater than 95% of deacetylation. In other embodiments, chitosan may be blended with other synthetic polymers and proteins where desirable to provide additional functionality to a medical device or implant.
 The solvent system employed in the present invention is a non-aqueous solvent system comprising one or more non-aqueous solvents that can readily dissolve the biopolymer being coated. In one embodiment, the solvent system for depositing a chitosan-based coating is propylene carbonate (PC) and ethylene glycol (EG) at a ratio of 1:4 v/v. For a two- or three-dimensional chitosan crystal morphology, a higher proportion of EG is employed. In another embodiment, the electrolyte further comprises a supporting electrolyte for improving control of the electrical potentials being applied by the electrodes. A supporting electrolyte, as known in the art, is a chemical species that is not electroactive at the electrical potentials used during electrochemical deposition, and has an ionic strength and conductivity much larger than those species that are electroactive in the electrolyte. Examples of the supporting electrolytes applicable to the present invention include, but are not limited to, LiClO4, KCl, KNO3, HCl, NaOH and various tetraalkylammonium salts. The concentration of the supporting electrolyte present in the electrolyte solution may be approximately 0.1-1.0 mol/kg.
 In one embodiment of the present invention, the electrolyte solution of the present invention is acidic. In other embodiments, the present method further comprises adjusting pH of the electrolyte by adding sufficient amounts of acids or bases. In one embodiment, sulphuric acid (H2SO4) is added as a pH adjusting agent. In one embodiment, the pH of the electrolyte of the present invention is adjusted to a pH of less than 6.3. In a preferred embodiment, the pH of the electrolyte applicable to the present invention may be adjusted to a in the range of 4 to 6.3, or more preferably to a pH of 4.5 to 6.3.
 The electrical potential and current applied in the present invention may be varied according to the desired morphology of the final biopolymer coating and the material being applied. An exemplary range of electric potentials applicable to the present invention are ranges that are less than approximately 10V. In certain embodiments, the electric potential may be less than 5V, or more preferably less than 1V. The electric current of the present invention has a current density of no more than on the order of less than 103 mA/cm2. In one embodiment, the current density is 103 mA/cm2.
 The present method further comprises incorporating at least one functional additive to the electrolyte for addition of functional properties to the resultant biopolymer coating. One of the technical features of the present invention is that functional additives can be readily incorporated into the biopolymer coating for additional function properties to facilitate the medical application of the instrument being coated, such as providing a therapeutic effect on the subject implanted with the medical device or treated using the medical instrument. The morphology and application of the biopolymer can also be varied to facilitate the desired medical application. For example, 5-florouracil is added into the electrolyte for providing an anti-cancer property to the biopolymer coating on said instrument (Example 1); calcium carbonate is added to the electrolyte for promoting cell proliferation in those cells or tissues in contact with the device or instrument (Example 2). Other examples of functional additives that can be employed in the present invention include, but are not limited to, therapeutic agents used singly or in combination for treatment, prevention or reduction of various diseases and conditions, for example, anti-thrombotic agents, anti-inflammatory agents, anesthetic agents, anti-coagulants, cell growth promoters, hormones, etc. The amount of a functional additive generally does not affect the deposition rate or morphology of the biopolymer coat. Where one or more types of functional additives are present in the coating prepared by the method of the present invention, the coating may comprise, for example, from 1 wt % or less to 2 wt % to 5 wt % to 10 wt % or more of the functional additives.
 Surfaces or articles that may be coated by the method of the present invention are typically electrically conductive or are modified to be electrically conductive. The conductive materials for said surfaces or articles include the following: (a) substantially pure metals, including gold, platinum, palladium, iridium, osmium, rhodium, titanium, zirconium, tantalum, tungsten, niobium, ruthenium, alkaline earth metals (e.g., magnesium), iron and zinc, and (b) metal alloys, including iron- and chromium-based metal alloys (e.g., stainless steels, including platinum-enriched radiopaque stainless steel), niobium alloys, titanium alloys, nickel alloys including nickel- and titanium-based metal alloys (e.g., Nitinol), cobalt- and chromium-based alloys, cobalt-, chromium-, and iron-based alloys (e.g., elgiloy alloys--that is, Co--Cr--Ni alloys), nickel-, cobalt-, and chromium-based alloys (e.g., MP 35N), cobalt-, chromium-, tungsten-, and nickel-based alloys (e.g., L605), and nickel- and chromium-based alloys (e.g., inconel alloys), and biodegradable metal alloys such as metal alloys where the main constituent thereof is selected from alkali metals, alkaline earth metals, iron, and zinc, for example, metal alloys containing magnesium, iron or zinc as a main constituent and one or more additional constituents selected from the following: alkali metals such as Li, alkaline-earth metals such as Ca and Mg, transition metals such as Mn, Co, Ni, Cr, Cu, Cd, Zr, Ag, Au, Pd, Pt, Re, Fe and Zn, Lanthanides such as La and Ce, Group 13 metals such as Al, and Group 14 elements such as C, Si, Sn and Pb.
 In one aspect of the present invention, the article is a medical device or instrument to be coated with a biocompatible material. Examples of medical devices that benefit from the biopolymer-containing coatings of the present invention vary widely and include implantable or insertable medical devices, for example, stents (including coronary vascular stents, peripheral vascular stents, cerebral, urethral, ureteral, biliary, tracheal, gastrointestinal and esophageal stents), catheters (e.g., urological catheters or vascular catheters such as balloon catheters and various central venous catheters), guide wires, balloons, filters (e.g., vena cava filters and mesh filters for distil protection devices), stent coverings, stent grafts, vascular grafts, abdominal aortic aneurysm (AAA) devices (e.g., AAA stents, AAA grafts), vascular access ports, dialysis ports, embolization devices including cerebral aneurysm filler coils (including Guglilmi detachable coils and metal coils), septal defect closure devices, myocardial plugs, patches, pacemakers, leads including pacemaker leads, defibrillation leads, and coils, ventricular assist devices including left ventricular assist hearts and pumps, total artificial hearts, shunts, valves including heart valves and vascular valves, anastomosis clips and rings, cochlear implants, tissue bulking devices, and tissue engineering scaffolds for cartilage, bone, skin and other in vivo tissue regeneration, sutures, suture anchors, tissue staples and ligating clips at surgical sites, cannulae, metal wire ligatures, urethral slings, hernia "meshes", orthopedic prosthesis such as bone grafts, bone plates, fins and fusion devices, orthopedic fixation devices such as interference screws in the ankle, knee, and hand areas, tacks for ligament attachment and meniscal repair, rods and pins for fracture fixation, screws and plates for craniomaxillofacial repair, artificial ligaments, joint prostheses, dental implants, or other devices that are implanted or inserted into the body.
 Using the methods, materials and conditions as described herein, highly uniform distribution of single crystal biopolymer coatings on irregularly shaped surfaces can be achieved. Moreover, different morphologies or nanostructure of biopolymer crystal can be achieved in accordance to the present invention by varying electrodeposition conditions. The terms "morphology" and "nanostructure" as used herein, are used interchangeably, refer to the form and structure of the single crystal of biopolymer coating deposited according to the present invention. Said form and structure may be one-dimensional, two-dimensional or three-dimensional and nano-sized ranging from 0.1-100 nm in each spatial dimension. Different morphologies of biopolymer crystals can be fabricated by the present method of electrochemical deposition. The morphologies resulted from the present invention can be, but are not limited to, particles, wires, rods, flakes, ribbons, flower-like, urchin-likes shapes, multipods and tubes.
 The present invention provides an electrochemical deposition approach for depositing a biocompatible material onto a surface in need thereof, and thereby forming a coating of said biocompatible material on the surface. The deposition of a material that occurs upon the application of an electrical potential between two conductive materials (electrodes) immersed in a non-aqueous liquid medium containing the material to be deposited (electrolyte). In various embodiments of the invention, a biopolymer is electrodeposited at the positive working electrode (i.e., the cathode where reduction takes place). A typical apparatus for carrying out electrodeposition includes the following: negative counter electrode (anode), a positive working electrode (cathode) and, optionally, a reference electrode, each separated by and in contact with an electrolyte (e.g. biopolymer containing solution). Electrodeposition can be carried out under a variety of electrochemical conditions including the following, among others: (a) constant current, (b) constant voltage, (c) varying pH, (d) varying solvent systems, (e) optional supporting electrolyte(s) and (g) a combination thereof, resulting in different morphologies of biopolymer crystals deposited on the positive working electrode. Furthermore, one skilled in the art would readily appreciate that the electrochemical deposition time of the present invention is varied according to the desired nano-sized of the biopolymer crystal and the electrochemical deposition can be repeated for multiple times for varied thickness of the biopolymer coating.
Electrodepositing A Chitosan Coating Containing 5-Florouracil Onto A Stainless Steel Surface
 To demonstrate electrodeposition of chitosan coatings incorporating 5-florouracil (5-FU) onto a stainless steel surface according to the present invention, the chitosan and 5-florouracil containing electrolyte is prepared by dissolving 8 mg of chitosan in 45 ml PC:EG (1:4 v/v). An appropriate amount of H2SO4 is added to adjust pH of the electrolyte to pH 4-4.5. The electrolyte is then subject to ultrasonication for 2 hours for proper dissolution. Supporting electrolyte LiClO4 at 5.5 mM or 55 mM, respectively along with 65 mg 5-florouracil are added to the acidic electrolyte. In this example, the ratio of chitosan to 5-FU is 1:8. Stainless steel surface to be coated with the chitosan and 5-FU and the prepared acidic electrolyte are set up according to FIG. 1 and undergo electrochemical deposition at -5V for 40 mins.
 FIGS. 2A-D show that a 3-dimensional flower-like morphology and an increased concentration of supporting electrolyte result in a denser chitosan deposition on the stainless steel surfaces. This is due to the enhanced ionic conductivity provided by a higher concentration of supporting electrolyte which increases the deposition rate.
 HeLa cell viability on a chitosan 5-florouracil coating on the stainless steel surface prepared according to Example 1 is then tested to demonstrate that the present method of coating maintains the functionality of the incorporated functional additive. Curve (a) in FIG. 3 shows that HeLa cells continue to grow and populate on bare (uncoated) stainless steel surfaces. On the other hand, coated stainless steel (curves (b) to (e)) all undergo an initial drop in cell viability indicating that chitosan coatings may initially suppress proliferation of HeLa cells. Only stainless steel coated with dense chitosan and 5-florouracil can impose a sustained tumor-killing effect. The sustained tumor-killing effect of the dense chitosan/5-florouracil coating reflects that the higher amount of 5-florouracil can be incorporated therewith as compared to the sparse coating. The results demonstrate that functional additives can be easily incorporated into biopolymer coatings, such as chitosan, using the present method of coating, without destroying the inherent functional properties of the additives themselves. The results also demonstrate that the present invention can be used as a drug packaging/delivery technique (e.g. via the chitosan coating).
Electrodepositing A Chitosan Coat With Calcium Carbonate (CaCO3) Onto Titanium Surface
 The same conditions and procedures as illustrated in Example 1 are applied to electrodeposit chitosan coatings with CaCO3 onto a titanium surface. Instead of florouracil, 40 mg of CaCO3 and 55 mM LiClO4 are added to the electrolyte solution. The ratio of chitosan to CaCO3 is 1:5. The morphology of chitosan coating having CaCO3 incorporated therein prepared according to the present method is observed and MC3T3-E1 cell viability on bare titanium versus coated titanium is tested. The results show that not only does the addition of CaCO3 to the chitosan can significantly promote cell adhesion and proliferation as compared to bare titanium surface and the chitosan coating alone (FIG. 4B), the CaCO3 incorporated chitosan coating is also observed to form a nanoflake morphology on the titanium surface under the SEM (FIG. 4A). The results in this Example further demonstrate that the present method of coating results in different morphology of chitosan coatings if different functional additives are added, and the functional properties of the additives is preserved using the present method.
 In view of the above exemplary embodiments, one skilled in the art would readily appreciate that simply by varying composition of the electrolyte, pH thereof and electric field applied thereto can easily adopt different morphology and density of biopolymer coat. It is also appreciated that present method of coating can be readily modified by one skilled in the art to tailor make a chitosan coat of specific characteristics suitable for the instrument being coated with.
 It is understood that the methods described herein may be performed in different orders, concurrently and/or together with other steps not mentioned herein but readily appreciated by one skilled in the art to obtain a chitosan or other biopolymer coating. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, modify the present invention without departing the spirit of the present invention and utilize the present invention to its fullest extent as set forth in the appended claims. All publications recited herein are hereby incorporated by reference in their entirety.
 The present invention discloses a novel method of coating chitosan onto surfaces in need thereof. The present method demonstrates that the morphology of the resulting chitosan coating can be highly controlled and demonstrates the ease of incorporating functional additives into a chitosan coating without altering the functional property of the additive. The resulting coating of chitosan by the present method also enhances the therapeutic effect of the incorporated functional additive by extending the release profile of the functional additive. Furthermore, as exemplified herein, the present invention provides a convenient channel for drug packaging/delivery, for example, onto implantable or insertable medical devices. In view of the foregoing advantages and characteristics associated with the present invention, one skilled in the art would readily knowledge the application of the present method in coating a wide range of device, instruments and equipment in need thereof.
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