METAL NANOPARTICLES IN A CHITOSAN MATRIX

PRIORITY

This application claims priority to U.S. Provisional Application No. 61 307,648, filed with the U.S. Patent and Trademark Ofifice on February 24, 2010, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to an electrochemical reduction method for deposition of a nanoparticle/chitosan composite layer and, in particular, a method for dispersion of metallic nanoparticles within a chitosan matrix on a metallic surface.

2. Background of the Related Art

Metal nanoparticles act as catalysts in a variety of chemical processing methods, including conversion of organic compounds for use in energy generation such as polymer membranes for hydrogen fuel cells, chemical synthesis such as carbon-carbon bond formation, and oxidation reactions. Fabrication of catalytic metal nanoparticles that include Silver (Ag), Gold (Au), Platinum (Pt) and Palladium (Pd) typically involve a three stage process that requires a metal salt in solution; a shaping or encapsulation agent, which is usually an organic molecule such as chitosan; and a strong reducing agent to reduce metal ions for the formation of nanoparticles. For example, Huang, et al. (Colloids and Surfaces B: Biointerfaces, 39 (2004), pages 31-37) discloses metal-chitosan nanocomposites through reduction of Ag, Au, Pt and Pd salts in the presence of chitosan through exposure to sodium borohydride, as a rapid process. However the third stage is highly reactive and can create an environmental or health hazard.

Chitosan is a linear polysaccharide of 2-amino-2-deoxy-D-glucopyranose obtained by deacetylation of chitin from crustaceans, mollusks, insects or fungi. Chitosan is the second most abundant natural biopolymer and there are broad ranges of applications for Chitosan. Chitosan, as well as chitosan loaded with antibiotic such as gentamicin, are biocompatible and have been applied to stainless steel bone screws to inhibit bacteria growth.

While biomedical and pharmaceutical applications have been exploited for some time, potential uses of chitosan-based biomaterials in industry are hindered by questions of stability, variability in properties, and production considerations. Surfaces for flexible electronics, sensor surfaces and device development require polymeric materials having elasticity as well as toughness and environmental durability.

In particular, durability and mechanical toughness, as well as adhesion to metal substrates, are challenges to applications that utilize chitosan. For example, silane coupling agents are needed to create a bond between a chitosan film and titanium substrates used in dental implants. This requires a complex process involving several chemical treatments, including curing at elevated temperature, reaction with a cyano-oxysilane coupling agent and overnight exposure to a glutaraldehyde cross-linking agent. Such processes and agents are undesirable and often environmentally unsafe.

An example of an environmentally unsafe method is provided by Huang et al.

(Colloids and Surfaces A: Physicochem. Eng. Aspects 226 (2003), pages 77-86), which describes techniques for incorporation of Au nanoparticles in a chitosan matrix. Huang requires pre-forming of the Au particles and stabilizing using citrate in a strong acidic solution prior to incorporation in chitosan, and also requires use of glutaraldehyde as a cross-linking agent. Huang et al. (Journal of Colloid and Interface Science 282 (2005), pages 26-31) describes a process, which requires use of sodium borohydride, which is a hazardous reducing agent, as does Adlim et al. (Journal of Molecular Catalysis A: Chemical 212 (2004), pages 141-149), in regards to obtaining Pt and Pd chitosan nanoparticles. Further, Huang et al. (Carbohydrate Research, 339 (2004), pages 2627-2631), describes a method for synthesizing Au and Ag nanoparticles, but requires elevated temperatures reaching 70 °C during the process. Raveendran et al. (Journal of the American Chemical Society 125 (2003), pages 13940-13941) attempts to provide an environmentally benign, i.e. "green", synthesis of Ag nanoparticles, but also requires elevated temperatures.

Accordingly, there is a need for an environmentally benign process for deposition of chitosan composite coatings via electrophoretic process, which does not require harsh reducing agents, elevated temperatures or non-green solvents.

SUMMARY OF THE INVENTION

The disclosed method overcomes the above shortcomings by providing a method for electrochemically-induced deposition of a submersed stainless steel electrode in an acidic chitosan solution with a predetermined concentration of cationic silver by applying a potential to the stainless steel electrode for a predetermined amount of time, coating an oxy- anion rich passive layer of the stainless steel electrode with a first silver / nitro-chitosan layer, and continuing coating of the electrode to form a subsequent semi-crystalline silver / chitosan layer, and an apparatus developed from such method.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of certain exemplary embodiments of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 illustrates electrochemical deposition on a cathode in accordance with the present invention;

FIG. 2 is a Scanning Electron Microscope (SEM) image showing distribution of silver (Ag) nanoparticles within a chitosan coating adhered on stainless steel substrate utilizing the method of the present invention;

FIG. 3 is a SEM image showing Ag nanoparticles formed near a surface of a chitosan layer deposited on stainless steel according to the method of the present invention;

FIG. 4 is a profile view of a chitosan-based coating applied to stainless steel utilizing the method of the present invention; FIG. 5 charts data from X-ray Absorption Near Edge Spectroscopy (XANES) of X- ray absorption energy versus intensity of an Ag foil standard and of Ag nanoparticles formed in chitosan on stainless steel according to the method of the present invention;

FIG. 6 shows Synchrotron Fourier Transform Infrared (FTIR) spectra of a chitosan- Ag nanoparticle composite coating obtained from the method of the present invention and of a pure chitosan coating;

FIG. 7 provides comparative graphs of synchrotron Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy of an Ag nanoparticle containing chitosan coating to an Ag foil standard;

FIG. 8 illustrates a process utilized to assess bonding strength of chitosan

electrochemically deposited according to the process of the present invention; and

FIG. 9 is a flowchart summarizing a method for electrochemically-induced deposition on an electrode according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A description of detailed construction of preferred embodiments is provided to assist in a comprehensive understanding of the exemplary embodiments of the invention. Those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the invention. Descriptions of well-known functions and constructions are omitted for clarity and conciseness.

The present invention provides a rapid technique utilizing room temperature solutions for electrochemical reduction to develop a nanoparticle/chitosan layer composite. Electrochemical deposition allows for use of a reduced number of metal ions in the design and development of composites, with the entire process performed in an environmentally friendly manner. As explained herein, an electrochemical method for polysaccharide attachment and film growth is provided via a negatively charged surface and an electron transfer process to generate the polysaccharide layer on a passivated stainless steel surface, similar to formation of biofilms by bacteria during biofouling. See U.S. Publication No. 2005/0154361 Al as a background regarding a chitosan-metal complex, the contents of which are incorporated herein by reference.

As known in the art, providing an elevated charge to stainless steel cathode 101 provides electrons that bind to the protons (hydrogen ions) and hydrogen (H ₂ ) gas bubbles off, further to Equation ( 1 ):

2H ⁺ + 2e -» H ₂ (1)

In a preferred embodiment, chitosan is electrophoretically deposited on stainless steel, primarily by applying a voltage to generate an elevated pH, preferably greater than 6.3, adjacent to the surface of cathode 101, as well as by electrostatic attraction of cationic chitosan from the solution to the cathode surface. FIG. 1 illustrates coatings being deposited utilizing the room-temperature solution via electrostatic attraction. As shown in FIG. 1 , localized, near-surface changes in pH are created by polarization of the stainless steel substrate, i.e. cathode 101. A strong surface adhesion occurs due to association of chitosan functional groups with chromate and other oxyanions in a passive layer 109 adjacent to the stainless steel substrate 101, with deposition performed under normal atmospheric conditions. A chitosan film develops on the stainless steel substrate 101, with a pH gradient 107 elevating to above 6.3 as distance to the stainless steel substrate 101 decreases.

Applied voltage provides a rapid, simple way to form metallic nanoparticle structures in chitosan, which has applications in device fabrication and controlled surface biological and chemical effects. Metallic nanoparticle spatial distribution in the chitosan coating, i.e. a matrix of chitosan film formed on the surface of the electrode, is preferably controlled through the processing methodology, which allows for patterned deposition. Patterned deposition is obtained by application of pulsed voltage, thereby creating a layered structure. The varied control of processing parameters, including pulsing of the deposition potential, produces the layered structure containing silver (Ag) nanoparticles shown in FIG. 2, which is a Scanning Electron Microscope (SEM) image obtained by an ion beam- machined sample, focused on a two micron cross section area of a coating obtained by the method of the present invention. A passive film on stainless steel includes an inner layer of kinetic metal oxide barriers and oxyhydroxides, and an outer layer enriched in oxyanions. Since

polysaccharides, including chitosan, are known to bind to chromate and other oxyanions in solution, an initial chitosan layer is created through electrostatic interaction with a cathodically charged stainless steel surface. In a preferred embodiment, a type 304 stainless steel is utilized, the composition of which is known in the art. After creating the Ag-chitosan matrix, a phenomenon of heightened adherence, which includes an improved mechanical strength, was observed based on inclusion of Ag nanoparticles, potentially further improved due to association of Chromium (Cr) (VI) with amine groups. This phenomenon contributes to initial film formation and enhances mechanical properties, including adhesion.

As described above, an electrochemically-induced process is preferably used to deposit a chitosan/metallic nanoparticle coating on a stainless steel, functioning as an electrode. In initial studies, a chitosan hydrochloric acid (HC1) solution having a pH of 4.5 to 5.0 was used. A 300 series stainless steel was found to provide a preferred reactive surface for deposition due to a passive layer that allows strong film adhesion. Deposition on type 304 stainless steel (18% Cr, 8% Nickel (Ni), bal. Iron (Fe)) at a cathodic potential was found to be rapid, with a thick layer of approximately 2-10 microns developing within five seconds to five minutes.

The coating method for electrochemically-induced deposition on an electrode submerses the electrode in an acidic chitosan solution with a predetermined concentration of cationic Ag. A volume of 50 ml of a 0.001 to 0.1 M solution of silver nitrate (AgN0 ₃ ) was added to 50 ml of a solution containing 0.5 g of low molecular weight chitosan and 0.5 ml of 50% by volume acetic acid solution to provide a solution for electro-deposition. A potential of -1.2 to -1.5 volts Ag/AgCl half cell potential was applied to the electrode. An

Ag chitosan coating having an Ag nanoparticle size varying from 5 to 100 nm rapidly formed.

Cathodic polarization of a stainless steel surface in a mildly acidic chitosan solution results in formation of an adherent and functionally graded process. Cationic chitosan is attracted to the cathodically-polarized surface where an initial, strongly bound layer is formed through complexation between amine (N¾) groups and chromate oxyanions in an outermost layer of the passive film. Through a deprotonation mechanism, chitosan is deposited from solution due to the pH gradient near the stainless steel electrode surface. Trapped hydroxyl radicals generated by the cathodic process oxidize C-OH and amine groups to form carbonate-like and nitrate-like functionalities.

After the cathode is removed from the solution, Ultra- Violet (UV) light exposure is preferably applied to dry the coating and further enhance the reactivity of hydroxyl radicals with chitosan, resulting in additional nitro groups. The dried coating develops with additional beneficial mechanical and adhesive properties, and is believed to enhance crystallinity by multiple forms within a functionally-graded structure. Introduction of a dilute noble metal ion to the solution, such as from dissolution of an Ag salt, facilitates growth and retention of stable metal nanoparticles for biomedical, catalysis, sensor and other applications such as water filtration and nuclear test containment is possible. The method of the present invention provides a durable coating with an improved mechanical durability for interfacing with other materials. In a preferred embodiment, a deposited Ag

nanoparticle/chitosan composite provides an anti-biofouling coating.

Fourier Transform Infrared (FTIR) and Raman spectroscopy were used to provide chemical analysis of runctionalized polysaccharide nanostructured materials and coatings. The Raman spectra from the electrochemically deposited coatings indicate a higher intensity in the primary amine bands, and occasionally in the phenolic region, as compared to stock powder. X-ray Photoelectron Spectroscopy (XPS) was utilized as a surface sensitive technique to analyze C, N and O speciation and chemical environment to a depth of approximately 10 nm, to confirm surface chemistry.

FIG. 3 is an SEM image showing Ag nanoparticles formed in the chitosan coating deposited electrochemically on stainless steel, and FIG. 4 is a profile view illustrating a structure obtained at the cathode by electrochemical deposition of a chitosan layer on the stainless steel electrode 101. Preferably the electrode contains Cr, as in stainless steel 304, or Cr and Molybdenum (Mo), as in stainless steel 316. As shown in FIG. 4, an Ag/chitosan coating is deposited on a Cr ₂ Mo bearing stainless steel surface 401 having an oxy-anion rich passive layer 403, including an Ag/nitro layer 405, a semi-crystalline Ag/chitosan layer 407, and a functionalized surface 409.

By introducing AgN0 ₃ with concentrations ranging from 0.00 ImM to ImM to an acetic acid/chitosan solution, high concentrations of Ag nanoparticles ranging in size from 5 to 100 nm formed within three to ten seconds, based on overall solution pH and chemistry and electrochemical potential during deposition. As in the case of the electrophoretically deposited coating on stainless steel described above, UV radiation exposure is preferably used to expedite drying of the coating and enhance coating durability.

FIG. 5 is a graph of synchrotron X-ray absorption data comparing a silver foil standard 502 and silver nanoparticles 504 formed in chitosan on stainless steel, with the comparison indicating the metallic nature of the particles.

FIG. 6 provides results of Synchrotron FTIR spectroscopy performed on pure chitosan 602 and an electrochemically-formed layer with Ag nanoparticles 604, revealing several distinct differences, including the loss of a shoulder at wavenumbers 3440 (Al , A2) and replacement of the doublet at wavenumbers 1660/1590 by a single dominant peak at wavenumber 1600 (A3), both indicative of complexation at the amine group of chitosan. Additional changes occur in the peaks at wavenumbers 1300-1450 in the amide II region indicating additional complexation.

A further embodiment includes a tailored anti-microbial coating for cell scaffold applications. To test the electrochemically-formed Ag nanoparticle containing coating, a biological protocol was conducted by sterilizing Ag-chitosan coated substrates by immersing in 70% ethanol for two hours, after which the substrates were rinsed three times with sterile Phosphate Buffered Saline (PBS), and immediately transferred to a sterile tissue culture dish. The coatings were retained on the surface and appeared to be stable following treatment. A cell suspension consisting of murine pre-osteoblasts (MC3T3-E1) was seeded onto the Ag-chitosan substrates at a density of 5,000 cells per square centimeter. The cells were maintained in alpha Minimum Essential Medium (a-MEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After five days of incubation at 37°C (5% CO2, humidified), the samples were fixed with 3.7% formaldehyde and stained with 4',6-diamidino-2-phenylindole (DAPI) for nuclei visualization. Immunofluorescence micrographs were captured using a reflection microscope (Olympus 1X71) with a DAPI filter cube.

Ag nanoparticles formed in the chitosan-based coating are stable for at least six months under general indoor atmospheric conditions of temperature and humidity, unlike Ag nanoparticles formed through simple wet chemical processes in chitosan. i.e. by use of chemical reductants, which reoxidize and agglomerate in less than 24 hours. The Ag/ chitosan coating was also found to remain stable on a 304 stainless steel coupon following sterilization.

Microscopic analysis did not reveal cell growth on the Ag-containing composite. Furthermore, SEM Energy Dispersive Analysis by X-rays (ED AX) analysis showed only non-living organic residue from the solution and no cell growth, indicating that the coating is anti-microbial, particularly useful for coating for biomedical equipment and implants. Testing of a second sample of chitosan coating without Ag nanoparticles revealed some osteoblast growth and attachment. Hence by controlling Ag-nanoparticle incorporation and distribution, coatings are obtained that prohibit cell growth for scaffolds and act as anti- microbial surfaces for biomedical instruments and devices.

To determine the chemical nature of the bonding layer created by the deposited matrix, stainless steel coupons 810 with deposited chitosan were immersed in liquid nitrogen for one minute. A razor was used to separate a large portion of the chitosan from the steel surface. Separated layers are shown in FIG. 8. Both the thin layer of deposited material, which remained strongly adhered to the surface 802 and the underside 804 of the removed layer, were observed to have undergone initial chemical analysis by Raman and X-ray Photoelectron Spectroscopy (XPS). FIG. 8 also provides spectra of the surface 802 and underside 804, which is found consistent not only with expected chitosan functional groups and bonding, but also consistent with formation of oxidized carbon and nitrogen species. A photoelectron spectra 805 obtained form underside 804 of the razor-removed coating, indicates formation of nitro and carbonyl groups.

The observed formation of such nitro groups supports a mechanism of formation and remarkable mechanical properties generated by the deposited matrix. The oxidation of amino (N ^3' ) to nitrate-like (N ⁵⁺ ) groups is typically only possible under rather extreme conditions, and in the presence of a strongly oxidizing species, such as hydroxyl radicals. This formation of reactive hydroxyl radicals, which react between the initially bound layers of the coating and the subsequent gel-like layers of deposited chitosan, plays a significant role in development of the structure, chemistry and properties noted of the coating obtained by preferred embodiments of the present invention. A Raman Spectra 803 obtained from surface 802 of the stainless steel indicates residual amine and nitro-enriched chitosan.

Further mechanical testing of electrochemically-deposited pure chitosan coatings indicated a coefficient of friction between 0.16 and 0.27, and elasticity of these coatings was found to range from 5-7 gigapascals (GPa).

FIG. 9 is a flow chart summarizing the coating method for electrochemically- induced deposition on a stainless steel electrode of the present invention. In step 901, a stainless steel electrode is immersed in an acidic chitosan solution, preferably including 0.001 to 0.1 M AgNC>3. The acidic chitosan solution is preferably 1 gram of low molecular weight chitosan in 100 mL of deionized water with an additional 0.5 mL of a 50% by volume acetic acid solution, which provides a pH of between 4.5 and 5.0. In step 903, a potential of -1.2 to -1.5 V versus the Ag/AgCl half cell potential is applied for a

predetermined amount of time. The predetermined amount of time is preferably between five seconds and five minutes.

In step 905, the stainless steel electrode is coated with an Ag/nitro-chitosan layer on an oxy-anion rich passive layer of the stainless steel electrode. In step 907, the coating continues to form a semi-crystalline Ag/chitosan layer. The Ag nitro-chitosan layer and the semi-crystalline Ag/chitosan layer being 2-10 microns thick and including 5-100 nm Ag particles. In a preferred embodiment, the stainless steel electrode is removed from the acidic chitosan solution and exposed to UV light for ten minutes at 365 nanometers wavelength, and 15,000 μ /cm ² at a distance of 10 cm.

Using the methodology described above, samples are deposited on mechanically polished 304 and 316 stainless steel coupons. The two metal substrates are used both to examine the role of substrate composition (Cr in 304 versus Cr and Mo in 316) on coating adhesion and interfacial chemistry. Both the 304 and 316 coupon types are commonly used in biomedical and other applications. The 304 stainless steel is a widely used austenitic stainless steel. The 316 steel is a common Mo-bearing austenitic stainless steel. Both 304 and 316 type steel are used extensively in medical devices, instruments and implants for energy applications, including transport lines, fuel cell components, and support surfaces for catalysts, as well as to provide structural support in electronics and for water treatment applications.

The polished coupons were approximately one square centimeter in size, and were mechanically polished rather than electropolished, a process shown to sometimes alter surface chemistry. All samples were ultrasonically cleaned in propanol and doubly distilled water. Acetic acid-based chitosan solution was used for deposition as acetic acid is environmentally benign, easy to dispose of, and produces excellent coatings.

A PAR 600 potentiostat was used for electrochemical deposition, with voltage and time of deposition varied to optimize processing. Processing voltage was varied from -2.5 to -0.5 volts for a saturated Ag/AgCl electrode in 0.1 V increments. Processing time was varied from five seconds to two minutes. Electrode/sample geometry during deposition was standardized through use of a custom test stand to hold the surface, i.e. the working electrode, at a set distance from the reference electrode, i.e. the Ag AgCl half-cell and the Pt counter electrode.

Electrochemical solutions were varied in terms of concentration: concentration of chitosan/acetic acid varies for pure chitosan coating deposition, and concentration of chitosan/acetic acid and AgNC»3 varies for Ag-containing deposition. All deposition was conducted in open, i.e. aerated, solution.

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.