Antimicrobial Evaluation of Metal Microneedles Made by Local Electrodeposition-Based Additive Manufacturing on Metal-Coated Substrates

Electrochemical-based additive manufacturing of metals has many potential uses for the manufacturing of medical devices with small-scale features. In this study, we examined the in vitro antimicrobial properties of metal microneedles made by local electrodeposition-based additive manufacturing called CERES (Exaddon AG, Switzerland) on metal substrates. Three-by-three arrays of copper microneedles were created on copper-coated silicon substrates. To understand the effect of a galvanic couple between gold microneedles and a copper substrate on the antimicrobial activity of the microneedle device, three-by-three arrays of copper microneedles were created on gold-coated silicon substrates. Scanning electron microscopy was used to understand the microstructure of the microneedles; the microneedles were shown to possess hollow bores and sharp tips. X-ray photoelectron spectroscopy indicated the presence of copper, carbon, oxygen, silicon, and nitrogen as well as the absence of toxic impurities for the copper microneedles on copper-coated silicon substrates. X-ray photoelectron spectroscopy indicated the presence of copper, carbon, oxygen, copper, gold, and silicon as well as the absence of toxic impurities for the copper microneedles on gold-coated silicon substrates. The copper surface was noted to have Cu (II) oxide or hydroxide. In vitro cell colonization studies involving the gram-positive bacterium Staphylococcus epidermidis, the gram-negative bacterium Escherichia coli, and the opportunistic fungal pathogen Candida albicans at 2 h and 24 h colonization at 37°C showed generally stronger activity for copper microneedles on copper-coated silicon substrates than for copper microneedles on gold-coated silicon substrates and uncoated silicon substrates. The copper microneedles on gold-coated silicon substrates showed stronger antimicrobial activity than uncoated silicon substrates except for 24 h colonization with Escherichia coli. The results of this study show potential strategies for creating antimicrobial microneedles for medical applications via local electrodeposition-based additive manufacturing.


INTRODUCTION
Copper is used in several types of medical devices, including intrauterine devices for contraception purposes, because of its antimicrobial properties. 1,20][11][12][13][14][15][16][17][18][19][20][21]  Shende et al.  showed that copper nanoparticles possess inhibitory activity against fungi such as Fusarium culmorum, F. oxysporum, and F. graminearum. 22rass et al. previously described several possible mechanisms by which copper exhibits antibacterial and antifungal activity. 23One mechanism involves the generation of hydroxyl radicals via a Fentontype reaction; these radicals can oxidize lipids, proteins, and other molecules within cells. 24In addition, copper ions can react with sulfhydryl groups to generate hydrogen peroxide. 25Moreover, copper can displace iron in iron-sulfur clusters (e.g., isopropylmalate dehydratase and other iron-sulfur dehydratase enzymes) within cells. 26The binding of copper ions to domains with a negative charge on the bacterial cell membrane can result in membrane depolarization, membrane leakiness, and possibly membrane rupture. 27,28Several of these mechanisms have been observed in the interaction of enterococci with copper-containing surfaces; for example, Warnes et al. showed that DNA degradation, reactive oxygen species generation, and respiratory inhibition steps were rapid and occurred before cell membrane damage. 29In a subsequent study, Warnes et al. reported that rapid membrane depolarization and slow DNA degradation processes were associated with the exposure of E. coli and Salmonella to copper-containing surfaces. 30The antifungal activity of copper against Candida albicans is attributed to the binding of copper to phospholipids such as phosphatidylserine and phosphatidylethanolamine, which are located in the fungal plasma membrane. 31everal parameters determine the potency of copper materials, including the forming of the material (e.g., surfaces or nanoparticles), humidity of the environment, temperature, and presence of buffers or other chemicals. 30,32,33The contact killing mechanism is also believed to be dependent on these environmental parameters and the type of microorganism. 92][43][44][45][46][47][48][49][50][51][52] Cui et al. described mechanism of action of gold nanoparticles against multidrug-resistant gram-negative bacteria via proteomic and transcriptomic approaches. 53They described two mechanisms; one mechanism involves the inhibition of a ribosome subunit from binding tRNA; the other involves the collapse of the membrane potential and the inhibition of ATPase activities.Unlike copper nanoparticles, gold nanoparticles do not exhibit antibacterial activity via a reactive oxygen species (ROS)-dependent mechanism.Using a well diffusion method, Jayaseelan et al. showed that gold nanoparticles possess antifungal activity against Aspergillus flavus, Aspergillus niger, C. albicans, and Puccinia graminis tritci. 54fforts have been made to create galvanic couples between two metals to promote antibacterial activity; in this approach, the anode in the galvanic couple is corroded at a more rapid rate than an identical metal in an isolated arrangement. 55,568][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73] As a general consideration, a difference > 0.15 V in galvanic index should be sufficient to observe galvanic corrosion of the anode material for harsh environments (e.g., those containing humidity and/or salt such as those commonly encountered by medical devices).
One approach that is finding greater use in metal medical device manufacturing is additive manufacturing. 740][81] In particular, the additive manufacturing approach involves the ejection of a metal electrolyte from a 300-nm-diameter nozzle, called an ''iontip,'' which is integrated with an atomic force microscope cantilever.The cantilever contains microfluidic channels with features similar to those of FluidFM cantilevers. 82,83This approach is often used to create pure copper structures, because it is associated with a high Coulombic efficiency for a wide range of voltage values. 79The electrodeposition-based additive micromanufacturing approach is associated with several benefits over other methods, including processing under ambient conditions, processing with minimal waste, processing without using a template, and processing at a high rate (via optimization of applied potential and printing pressure parameters); in addition, this approach does not require post-processing steps. 84,85For example, Eliyahu et al. processed dense and uniform polycrystalline copper pillars with straight and overhang features that exhibited diameters of 1.5 lm-250 nm. 86Ercolano et al. showed that the lateral diameter of the copper voxels could be modified by modulating the applied pressure and the probe aperture. 818][89][90] For example, Sachan et al. prepared 431-lm-tall, hollow, copper microneedles 91 ; microneedle devices are lancet-or thorn-shaped devices that have potential use for the delivery of drugs and vaccines through the stratum corneum layer of the skin with less pain and damage than conventional methods. 92Hollow microneedle devices can be used for ''poke and flow'' delivery, in which diffusion or effusion of a drug or vaccine occurs through the bores of the hollow microneedles. 91According to Waghule et al., a microneedle used for skin penetration and drug delivery should exhibit a sharp and tapered tip, width over the 50-250 lm range, length over the 150-1500 lm range, and tip thickness over the 1-25 lm range. 93The tip of the microneedle can exhibit a wide variety of shapes, including cylindrical, pointed, and triangular. 94n recent years, there has been greater interest in (1) developing microneedles with antimicrobial properties to reduce the risk of skin infection associated with the microneedle penetration process and (2) delivering antimicrobial agents with microneedles. 95,96In one study, Gittard et al. used pulsed laser deposition to grow silver and zinc oxide coatings on the surfaces of polymeric microneedle arrays, which were created using visible light Digital Micromirror Device (DMD TM )-based dynamic mask microstereolithography.8][99] Agar diffusion studies were used to understand the antimicrobial activity of coated microneedle arrays.The agar diffusion studies showed that the silver and zinc oxide thin films gave the microneedle devices with antibacterial properties against Staphylococcus epidermidis and S. aureus.However, significant efficacy against E. coli was not observed; although the silver-coated microneedle arrays showed areas with an absence of growth, the zinc oxide-coated microneedle arrays showed zones of reduced growth.In contrast, the uncoated arrays showed no antimicrobial effect.This study indicated that pulsed laser deposition may provide acrylate-based polymer microneedle arrays with antimicrobial properties.
In this study, we consider the use of electrodeposition-based additive micromanufacturing to create metal microneedle arrays and the antimicrobial activity of these microneedle arrays against the gram-positive bacterium S. epidermidis, the gramnegative bacterium E. coli, and the opportunistic fungal pathogen C. albicans.Three-by-three arrays of copper microneedles were manufactured on copper-coated silicon substrates.The base was chosen to be larger to ensure decent adhesion to the substrates; the taper on the top was chosen to enable the printed structure to be in the form of a needle, which would be beneficial for insertion into skin or other tissue.From a sample manufacturing standpoint, the inclusion of a taper does not introduce any difficulty or significant change.A height of 500 lm was chosen for this specific application, which is well in 3D printable range for the electrodeposition-based additive micromanufacturing approach.Per Larran ˜eta et al., metals such as copper are suitable materials for manufacturing microneedles since they exhibit relatively high fracture toughness and Young's modulus values. 100o examine the effect of a galvanic couple between gold microneedles and a copper substrate on the antimicrobial activity of the microneedle device, three-by-three arrays of copper microneedles were manufactured on gold-coated silicon substrates.The microstructure of the microneedle arrays was evaluated by scanning electron microscopy, and the chemical composition of the microneedle arrays was examined using X-ray photoelectron spectroscopy.The results of these studies are considered in the context of potential medical device applications.

Manufacturing of the Microneedle Arrays
The microneedles were produced using the localized electrodeposition approach with a CERES instrument (Exaddon AG, Glattbrugg, Switzerland).This instrument contains a hollow atomic force microscopy cantilever, which is called an iontip, for transporting copper ions to the working electrode component of a three-electrode electrochemical cell.When a potential is applied to the working electrode, it enables the ions to be reduced to a dense metal.The CERES instrument processes material in a voxel-by-voxel manner; in this approach, the iontip is positioned above the substrate (or above previously deposited metal) until the growth of the metal touches the iontip, which causes the deflection of the cantilever.Measurement of this deflection is accomplished via an optical beam deflection approach, in which deflection prompts the instrument to relocate the iontip to a new voxel position.
A commercially available copper ink (Cu iontip ink, Exaddon AG, Glattbrugg, Switzerland) containing copper sulfate was used in this study.The electrochemical cell used in the printing approach was filled with a solution containing sulfuric acid (Cu bright printing solution, Exaddon AG, Glattbrugg, Switzerland); 1.5 cm 9 1.5 cm silicon wafers with 50-nm-thick copper or gold coatings were used as substrates in this study; a 16-nm-thick adhesion layer of titanium was deposited on the Si wafer prior to deposition of the copper or gold coating.
Electrodeposition-based additive micromanufacturing was used to process an array of nine copper microneedles, each with a height of approximately 500 lm, on each copper-or gold-coated substrate; the spacing between the needles was 250 lm.The microneedles were processed in sections of 100 lm because of design limitations imposed by the clearance of the iontip.Each section consisted of 100 voxels; each of these voxels exhibited a height of 1 lm.The average print time per voxel was 900-1000 ms, which indicates a print speed of 1 lm s À1 .The total duration required to print nine needles on each substrate was 75 min.
The diameter of the printed voxels (5 lm), and thus of the microneedles (since they are one voxel in diameter), was controlled by the pressure applied during the additive manufacturing process; the pressure applied to the ink reservoir determined the quantity of ions that was provided to the iontip.The tapered shape of the microneedle tip was obtained by decreasing the applied pressure for the last 80 voxels from 500 to 20 mbar.

Characterization of the Microneedle Arrays
Images of the microneedles were obtained with a Gemini Electron microscope (Carl Zeiss Microscopy GmbH, Jena, Germany); an acceleration voltage of 8 kV was used in this study.Measurements of the microneedles were obtained using a S-4700 microscope using an acceleration voltage of 2 kV; these samples were coated with a 10-nm-thick gold-palladium (AuPd) alloy coating prior to imaging.
The chemical composition of the surface of the copper microneedle arrays on copper-and goldcoated surfaces was assessed using X-ray photoelectron spectroscopy.Data were collected using a Supra + instrument (Kratos Analytical Inc., Nanuet, NY) that contained a monochromatic Ka X-ray source, which was operated at 150 W. A charge neutralizer was utilized to prevent charging; all of the spectra were corrected to the C 1 s peak at 284.8 eV.Survey scans were obtained at a pass energy of 160 eV; a spot size of 110 lm was used in this study.
The antimicrobial properties of the copper microneedle arrays on 1.5 cm 9 1.5 cm copper and gold coated surfaces as well as uncoated 1.5 cm 9 1.5 cm diced Si wafers were studied using an in vitro approach as described previously. 101Uncoated Si wafers and electrodeposition-based additive micromanufacturing-processed copper microneedle arrays on gold or copper substrates were assessed in this study.Two replicates for each surface were attached to the bottom of 100-mm Petri dishes with double-sided adhesive tape; the samples were then sterilized in a laminar flow hood by irradiation with ultraviolet light for 15 min.Fifteen milliliters of a 10 9 cells/mL suspension of E. coli (ATCC 12435, American Type Culture Collection, Manassas, VA) or S. epidermidis (ATCC 35984, American Type Culture Collection, Manassas, VA) was added to the surface of each sample, resulting in complete sample immersion; the samples were incubated statically at 37°C for 2 h or 24 h to allow for microbial colonization.The surfaces of the samples were rinsed three times with 15 mL of 1 9 phosphatebuffered saline with gentle swirling in between each rinse to remove unattached cells and aseptically transferred into 50 mL conical tubes, which contained 5 mL of Dey-Engley (DE) neutralization broth.The conical tubes were vortexed for 30 s, sonicated for 2 min, and vortexed again for 30 s to recover viable bacteria that were adhered to the surfaces of the samples.Each DE recovery solution was serial diluted ten-fold to a 10 À8 concentration; 10 lL of each dilution was subsequently pipetted onto Plate Count Agar (PCA) in triplicate.The PCA plates were incubated overnight at 37°C; the number of colonies was counted to evaluate the total number of viable cells recovered from the surface of each sample.

RESULTS AND DISCUSSION
Figure 1a and b shows scanning electron micrographs of the array of hollow copper microneedles at low and high magnification, respectively.Figure 1b indicates that the microneedles have sharp tips.Measurements taken from scanning electron micrographs of three microneedles flattened on the substrate indicate that the average microneedle length is 494.6 lm, which is similar to the height of the computer-aided design model used for microneedle manufacturing (500 lm).The microstructural features of the microneedles, including the sharpness of the microneedle tip, indicate that the devices may be useful for the transdermal delivery of drugs or vaccines.
Figure 2 shows the X-ray photoelectron spectra obtained with a 110-lm-diameter spot size from (1) an array of copper microneedles on a copper-coated substrate and (2) an array of copper microneedles on a gold-coated substrate.The survey scan of the array of copper microneedles on a copper substrate indicated the presence of carbon, copper, nitrogen, oxygen, and silicon; in contrast, the survey scan of the array of copper microneedles on a gold substrate indicated the presence of carbon, copper, gold, oxygen, and silicon.The presence of oxygen was associated with the formation of copper oxide on the copper surface; the presence of carbon was associated with the copper ink additive and the graphite electrodes used in the additive manufacturing process. 91No other elements, such as elements with known toxicity, were detected.This composition of the microneedle appears to be appropriate for transient contact with the skin.Figure 2c shows the Cu 2p line from an array of copper microneedles on a copper-coated substrate, and Fig. 2d shows the Cu 2p line of an array an array of copper microneedles on a gold-coated substrate; the shape of the Cu 2p line in these structures is consistent with a Cu (II) oxide or hydroxide.
Figure 3a and Table I(a) show the results of the cell colonization assessment with Staphylococcus epidermidis for 2 h colonization at 37°C; these data indicate that the array of copper microneedles on a copper-coated substrate provided greater anti-S.epidermidis activity (2.47 log reduction to uncoated silicon control) than the array of copper microneedles on a gold-coated substrate (1.30log reduction to uncoated silicon control).Figure 3b and Table I(b) show the results of the cell colonization assessment with S. epidermidis for 24 h colonization at 37°C; these data indicate that the array of copper microneedles on a copper-coated substrate provided greater anti-S.epidermidis activity (5.27 log reduction to uncoated silicon control) than the array of     oxide or hydroxide surface on the copper microneedles on the copper-coated substrate generally provided greater antimicrobial activity than the Cu (II) oxide or hydroxide surface on the array of copper microneedles on the gold-coated substrate.[40]

CONCLUSION
This study demonstrated that electrochemicalbased additive manufacturing can be used to create three-by-three arrays of copper microneedles on copper-coated silicon substrates and gold-coated silicon substrates.X-ray photoelectron spectroscopy showed that microneedle arrays contained no toxic impurities.In vitro cell colonization studies involving the gram-positive bacterium S. epidermidis, the gram-negative bacterium E. coli, and the opportunistic fungal pathogen C. albicans at 2 h and 24 h colonization showed generally stronger activity for copper microneedles on copper-coated silicon substrates than for copper microneedles on gold-coated silicon substrates and uncoated silicon substrates.As with other copper-and gold-containing medical devices, this activity can be attributed to damage to bacterial cell membranes and bacterial DNA.Additional studies are underway to better understand the mechanism of action associated with these devices, which will enable optimization of the antimicrobial activity of metal microneedle arrays for future applications in clinical medicine.

Fig. 1 .
Fig. 1.Images of an array of copper microneedles at (a) low and (b) high magnification.

Fig. 2 .
Fig. 2. X-ray photoelectron spectra with 110 lm spot size from (a) an array of copper microneedles on a copper-coated substrate, (b) an array of copper microneedles on a gold-coated substrate, (c) Cu 2p line from an array of copper microneedles on a copper-coated substrate and (d) Cu 2p line of an array an array of copper microneedles on a gold-coated substrate.

Table I .
Cell colonization assessment for the array of copper microneedles on a copper-coated substrate, the array of copper microneedles on a gold-coated substrate, and the uncoated silicon control Figure3fand Table I(f) show the results of the cell colonization assessment with C. albicans for 24 h colonization at 37°C; these data indicate that the array of copper microneedles on a copper-coated substrate provided greater anti-C.albicans activity (1.37 log reduction to uncoated silicon control) than the array of copper microneedles on a gold-coated substrate (0.58 log reduction to uncoated silicon control).These results indicate that the Cu (II)