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Overviewstructural, Functional, and Biological thin Films

deposition of antimicrobial coatings on microstereolithography-fabricated microneedlesShaun D. Gittard, Philip R. Miller, Chunming Jin, Timothy N. Martin, Ryan D. Boehm, Bret J. Chisholm, Shane J. Stafslien, Justin W. Daniels, Nicholas Cilz, Nancy A. Monteiro-Riviere, Adnan Nasir, and Roger J. Narayan

How would you……describe the overall signifi cance of this paper?In this manuscript visible light dynamic mask microstereolithography was shown to be an appropriate technique for scalable production of microneedle devices. The combination of dynamic mask stereolithography and pulsed laser deposition was used to create acrylate-based polymer microneedle arrays with antimicrobial properties, which may be used for treatment of local skin infections.…describe this work to a materials science and engineering professional with no experience in your technical specialty?Antimicrobial silver and zinc oxide thin fi lms were deposited on visible light dynamic mask microstereolithography-fabricated microneedles by means of pulsed laser deposition. The results of agar diffusion assays showed that the silver thin fi lms and zinc oxide thin fi lms provided the microneedle devices with antibacterial properties. Signifi cant effects against Staphylococcus epidermidis and Staphylococcus aures, two common pathogens, were noted.…describe this work to a layperson?Microneedles devices were fabricated in a layer-by-layer manner from computer models using a rapid prototyping process known as visible light dynamic mask microstereolithography. Antimicrobial silver and zinc oxide thin fi lms were deposited on the visible light dynamic mask microstereolithography-fabricated microneedles using a physical vapor deposition process called pulse laser deposition. These coated microneedle devices demonstrated signifi cant activity against two bacteria associated with skin infections, Staphylococcus epidermidis and Staphylococcus aures.

Microneedles are small-scale nee-dle-like projections that may be used for transdermal delivery of pharma-cologic agents, including protein-con-taining and nucleic acid-containing agents. Commercial translation of polymeric microneedles would benefi t from the use of facile and cost effec-tive fabrication methods. In this study, visible light dynamic mask microste-reolithography, a rapid prototyping technique that utilizes digital light projection for selective polymerization of a liquid resin, was used for fabrica-tion of solid microneedle array struc-tures out of an acrylate-based polymer. Pulsed laser deposition was used to de-posit silver and zinc oxide coatings on the surfaces of the visible light dynamic mask microstereolithography-fabricat-ed microneedle array structures. Agar diffusion studies were used to demon-strate the antimicrobial activity of the coated microneedle array structures. This study indicates that light-based technologies, including visible light dynamic mask microstereolithography and pulsed laser deposition, may be used to fabricate microneedles with antimicrobial properties for treatment of local skin infections.

introduction

Skin infections, including abscess, cellulitis, erysipelas, and impetigo, are an issue of growing medical impor-tance; for example, skin infections are associated with 7–10% of hospitaliza-tions.1,2 Gram-positive cocci bacteria (e.g., Staphylococcus aureus and group A streptococci) are most commonly as-sociated with infection of normal skin. A 2001 study indicated that the micro-organisms most frequently associated with skin infections were (arranged by

decreasing frequency) Staphylococcus aureus, Enterococcus species, coag-ulase-negative staphylococci, Esch-erichia coli, and Pseudomonas aeru-ginosa.3 Empiric antibiotic therapy for staphylococci and streptococci is the most common approach for skin infec-tion treatment; however, topical use of zinc oxide and silver may also fi nd use in skin infection treatment.4

There has been signifi cant interest in recent decades in the medical use of silver.5–7 As noted by Nadworny and Burrell, silver nitrate was introduced for burn treatment in 1965.8 In 1967, silver sulfadiazine cream was intro-duced for burn and wound treatment. Ag+ ions interact with sulfur-contain-ing proteins (e.g., proteins in bacterial membranes) and phosphorus-contain-ing compounds (e.g., DNA).5 Ag+ ions can also cause cell death by interact-ing with the respiratory chain within bacteria. Schaller et al. showed that silver-containing Contreet-H® wound dressings released silver and provided activity against methicillin-resistant Staphylococcus aureus and Candida albicans in an in vitro cutaneous in-fection model that contained reconsti-tuted human epithelium; furthermore, no major morphological effects were noted in treated epithelial cells.9 More recently, Bhattacharyya and Bradley demonstrated the use of nanocrystal-line silver (Acticoat 7®) for decreasing the spread of cutaneous necrosis and reducing methicillin-resistant Staphy-lococcus aureus loading in a clinical activity involving two patients.10 They suggested use of silver dressings as a “preemptive antimicrobial therapy” for management of diffi cult-to-treat and high-risk acute skin infections. Silver-containing ointments and moisturizers

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ExpErimEntal procEdurE

Figure A. (a) Layout of input STL files, which were used for fabrication of microneedle array structures by means of visible light dynamic mask micro-stereolithography. (b) Optical image of the microneedle array structures on the build platform. (c) Drawing of the input STL file of a mi-croneedle array structure; this structure consists of four microneedle arrays that are attached to the substrate.

a b c

A Perfactory III SXGA+system (EnvisionTEC GmbH, Glad-beck, Germany) was used to produce microneedle devices from a commercially available photosensitive acrylate polymer, eS-hell 200 (Envisiontec GmbH, Ferndale, MI), which is used in fabrication of thin-walled hearing aid shells.58 According to the manufacturer, eShell 200 is a Class-IIa biocompatible, water-resistant material. It contains 0.5–1.5% wt. phenylbis(2,4,6 trimethylbenzoyl)-phosphine oxide photoinitiator, 15–30% wt. propylated (2) neopentyl glycoldiacrylate, and 60–80% wt. ure-thane dimethacrylate. This acrylate-based polymer a exhibits a glass transition temperature of 109ºC (E1545-00 test method), a flexural strength of 103 MPa (D790M test method), a tensile strength of 57.8 MPa (D638M test method), and an elongation at yield of 3.2% (D638M test method); in addition, it exhibits a water absorption value of 0.12% (D570-98 test method). Previous nanoindentation studies provided hardness and Young’s modulus values of 93.8 ± 7.25 MPa and 3050 ± 90 MPa, respectively.59

The Perfactory III SXGA+ system (EnvisionTEC GmbH, Gladbeck, Germany) equipped with a Digital Micromirror Device SXGA+ (1280 × 1024-pixel resolution) guidance chip (Texas In-struments, Dallas, TX) and a halogen bulb was used for layer-by-layer polymerization of the acrylate-based polymer. Structures were fabricated within a 96.54 mm × 72.41 mm build envelope. Microneedle devices were fabricated at 550 mW using an expo-sure time of 3.5 seconds and a z-direction step size of 30 µm. Perfactory® RP software (Envisiontec GmbH, Ferndale, MI) was used to specify the layout of the microneedle devices in the build area. An image containing several input STL files is presented in Figure Aa. Structures were produced on the build platform ac-cording to the layout that had been specified using Perfactory® RP software. After fabrication, the build platform containing at-tached microneedle devices was removed from the resin-filled basin. An image of microneedle devices attached to the build platform, which corresponds to the layout shown in Figure Aa, is provided in Figure Ab. The microneedle devices were subse-quently removed from the build platform; support structures were also removed. The microneedle devices were then rinsed in iso-propanol (Fisher Scientific, Waltham, MA) and rinsed in acetone (Fisher Scientific, Waltham, MA). The microneedle devices were subsequently placed in an Otoflash Post Curing System instru-ment (EnvisionTEC GmbH, Gladbeck, Germany) for 50 seconds; this instrument provides light exposure over a 300–700 nm wave-length range for post-build curing. Five different types of structures were produced using the vis-ible light dynamic mask micro-stereolithography system in or-der to obtain resolution data. Two different test structures were produced to determine the hole resolution, which we will define as resolution of a hole produced in a 2 mm thick flat substrate.

The first hole resolution structure, shown in Figure Ba, is an ar-ray of circular holes having five replicates in the y-direction and in the x-direction; processing of 800 µm, 700 µm, 600 µm, 550 µm, 500 µm, 450 µm, and 400 µm diameter holes was attempted. The second hole resolution structure, shown in Figure Bb, is an array of square holes having 5 replicates in the y-direction and in the x-direction; processing of 800 µm, 700 µm, 600 µm, 550 µm, 500 µm, 450 µm, and 400 µm hole side-lengths was attempted. A third structure was produced to determine the line resolution of the visible light dynamic mask micro-stereolithography system; this structure is shown in Figure Bc. This structure consisted of lines with input widths ranging from 140 µm to 30 µm in 10 µm incre-ments; in this structure, gaps of 500, 300, 250, 200, and 150 µm were designed. After processing, the test structures were imaged with an EZ4 optical microscope (Leica Microsystems, Wetzlar, Germany) using LAS EZ software (Leica Microsystems, Wetzlar, Germany). Microneedle arrays with four different geometries were pro-duced using the visible light dynamic mask micro-stereolithogra-phy system. The heights and base geometries of these microneedle devices are shown in Table A. The microneedle devices exhibited rectangular pyramid shapes. Two different input pyramid heights, with heights of 1,000 µm and 1,250 µm, and two different base geometries, with dimensions of 500 µm × 250 µm and 750 µm × 250 µm, were produced. The tips of all four microneedle device geometries exhibited dimensions of 90 µm × 30 µm. Four identical 3 × 3 microneedle arrays were attached by support structures to a 1.0 mm thick and 18 × 18 mm substrate (Figure Ac).The STL de-signs for the microneedle devices were created using Solidworks software (Dassualt Systemes S.A., Velizy, France). The support structures were produced using Magics RP 13 software (Materi-alise NV, Leuven, Belgium). Eight substrates, each containing four microneedle devices, were produced in a single batch. Micronee-dles with heights of 1,000 µm and base dimensions of 750 µm × 250 µm were also produced on a cross-shaped substrate. Since porcine skin has similar morphology and thickness to hu-man skin, it is an acceptable model for human skin. A skin penetra-tion study was performed with full-thickness weanling Yorkshire skin. The ability of the microneedle devices to penetrate porcine skin was examined using trypan blue (Mediatech, Inc., Manassas, VA), a toluidine-based dye. The skin was stored at 3ºC until the skin penetration study was performed. A cross-shaped micronee-dle device was inserted into the porcine skin. Immediately after microneedle device removal, trypan blue was applied to the mi-croneedle insertion site. Residual dye was subsequently removed using isopropanol swabs. The microneedle devices were examined before and after porcine skin insertion using a Leica EZ4 optical microscope (Leica Microsystems, Wetzlar, Germany). Optical mi-

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Table A. Input Measurements and Scanning Electron Microscopy Measurements of Microneedle Dimensions

GeometryInput Height

(mm)

Actual Height (mm)

Input Width (mm)

Actual Width(mm)

Input Depth (mm)

Actual Depth (mm)

A 1,000 828 ± 19 750 805 ± 24 250 311 ± 17

B 1,000 665 ± 40 500 510 ± 9 250 285 ± 15

C 1,250 831 ± 9 750 817 ± 6 250 332 ± 21

D 1,250 748 ± 31 500 571 ± 25 250 347 ± 11

Figure B. Drawings of input files that were used for fab-rication of (a) circular holes, (b) square holes, and (c) line patterns. Optical images of (d) circular holes, (e) square holes, and (f) line patterns, which were fabricated by means of the visible light dynamic mask micro-stereo-lithography system.

a b c

d e f

croscopy was also used to examine the microneedle-fabricated, trypan blue-treated pores in skin. Pulsed laser deposition of silver thin films and zinc oxide thin films was performed us-ing a KrF excimer laser, which was operated at a wavelength of 248 nm, a pulse duration of 25 ns, and a repetition rate of 10 Hz. The coatings were deposited at room temperature and under 10–5 Torr vacuum for five minutes. For deposition of silver thin films, a 99.99% purity target was obtained from a commercial source (Alfa Aesar, Ward Hill, MA). For deposition of zinc oxide thin films, a 99.99% purity ZnO powder (Alfa Aesar, Ward Hill, MA) was pressed into round 2 inch diameter pellets and subsequently sintered at 900ºC in an oxygen atmosphere for 12 hours. Eight 250 × 500 µm base, 1,250 µm height mi-croneedle devices were coated with each of the two coating materials. Silver and zinc oxide thin films were also deposited on masked silicon wafers for profilometry studies. A Hitachi S-3200 scanning electron microscope (Hitachi, Tokyo, Japan) equipped with a Robinson backscattered electron detector was used to image the microneedle devices. The pulsed laser deposition-coated microneedle devices were imaged without further modification; the uncoated microneedle devices were sputter coated 60% gold-40% pal-ladium using a Technics Hummer II instrument (Anatech, Battle Creek, MI) in advance of imaging. A Dimension 3000 AFM with NanoScope analysis software (Veeco, Santa Barbara, CA) and an AC160 tip (Olympus, Melville, NY) operating in tapping mode was used to examine the surface topographies of pulsed laser deposition-grown silver and zinc oxide coatings on silicon substrates. A Tencor Alpha Step 200 stylus profilometer (PLA-Tencor, Milpitas, CA) was used to measure the thicknesses of the pulsed laser deposition-grown silver and zinc oxide coatings on silicon substrates. For each coated substrate, scans were performed in triplicate over a 400 µm range, which contained both coated and uncoated surfaces. A 20 mg stylus scanning at 10 µm/second was used for profilometry measurements. The antimicrobial activity of the pulsed laser deposition-coated microneedle devices was assessed using an agar diffusion assay. Escherichia coli ATCC 12435, Staphylococ-cus aureus ATCC 6548, and Staphylococcus epidermidis ATCC 35984 (American Type Culture Collection, Manassas, VA) were cultured overnight in tryptic soy broth (VWR International, West Chester, PA). The cell suspensions were then centrifuged at 4,500 rpm for 10 minutes. A cell suspension with a density of ~108 CFU/ml was then made by resuspending the pellet in 1x phosphate-buffered saline (VWR International, West Chester, PA). Mueller Hinton agar plates (VWR International, West Chester, PA) were inoculated with a lawn of bacteria using a sterile swab. The microneedle devices were placed on the inoculated plates with the coated side facing the agar; in this arrangement, the microneedle devices projected into the agar. After incubating at 37ºC for 24 hours, the surfaces of the plates were imaged with optical microscopy to evaluate microorganism-material interactions.

have also been developed for treatment of eczema.11

In addition to antimicrobial activ-ity, Jun et al. demonstrated that silver nanoparticles are associated with al-tered fibrogenic cytokine (e.g., VEGF, IL-10, and IFN-γ) levels as well as re-duced wound inflammation.12 Liu et al. examined topical application of silver nanoparticles in a murine full-thick-ness excisional wound model in mice; they showed that silver nanoparticles increase the wound closure rate by promoting migration and proliferation of keratinocytes into the wound bed.13 In addition, silver nanoparticles pro-mote differentiation of fibroblasts into myofibroblasts. Wright et al. examined the use of nanocrystalline silver-coated dressings in a porcine full-thickness wound model. Use of nanocrystalline silver-coated dressings was associated with rapid wound healing and reduced local matrix metalloproteinase levels; matrix metalloproteinases are believed to retard healing of chronic ulcers and other wounds.14 Elston noted that nano-crystalline silver is associated with lit-tle evidence of toxicity.15

Lansdown et al. noted that zinc-containing materials may be useful in wound care due to their activity against microorganisms that are commonly found in wounds.16 They stated that zinc inhibits growth of Gram-positive mi-croorganisms and Gram-negative mi-croorganisms; however, Gram-positive bacteria were noted to exhibit greater sensitivity to zinc than Gram-negative bacteria. Atmaca et al. examined zinc interaction with Staphylococcus au-reus, Staphylococcus epidermidis and Pseudomonas aeruginosa membranes; their work indicated that zinc prolongs the lag phase of the growth cycle.17 Sawai et al. noted that intracellular damage may be associated with pro-duction of hydrogen peroxide by zinc oxide.18 Zinc oxide may also directly damage the bacterial cell membrane, leading to intracellular content leakage and cell death.19 Disruption of Gram-positive bacterial membranes and Gram-negative bacterial membranes has been noted after zinc oxide expo-sure.20

Agren et al. and Akiyama et al. de-scribed zinc oxide-mediated inhibition of Staphylococcus aureus growth and

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coated with silver using pulsed laser deposition. The viability of human epi-dermal keratinocytes on silver-coated Ormocer surfaces was similar to that on uncoated Ormocer surfaces. The silver-coated Ormocer microneedles were shown to be effective at inhibit-ing growth of Staphylococcus aureus in an agar diffusion assay. In another study, two photon polymerization-micromolding was used to produce microneedle arrays out of a photosen-sitive material that contained polyeth-ylene glycol 600 diacrylate and 2 mg/mL gentamicin sulfate.39 The polyeth-ylene glycol 600 diacrylate- gentami-cin sulfate microneedles were shown to inhibit Staphylococcus aureus growth in an agar plating assay. In this study, microneedles were fab-ricated by means of a rapid prototyping process known as visible light dynamic mask microstereolithography. In rapid prototyping, structures are built up in a layer-by-layer manner from computer models. In this study, a commercial rapid prototyping system containing a Digital Micromirror Device (DMD™) was utilized for dynamic mask micro-stereolithography. In visible light dy-namic mask microstereolithography, a dynamic mask is used for selective polymerization of a photosensitive material. Since an entire layer of ma-terial is polymerized at once, process-ing times associated with visible light dynamic mask microstereolithography are significantly less than those as-sociated with other rapid prototyping processes. Through use of a system with a large build envelope, multiple structures can be built in parallel. Vis-ible light dynamic mask microstereo-lithography has been used to prepare several types of microscale biomedical devices. Sun et al. demonstrated use of Digital Micromirror Device-based mi-crostereolithography to create a micro coil array with a wire diameter of 25 mm and a coil diameter of 100 mm.40 In addition, they fabricated a high aspect-ratio micro rod array. In this study, a curing depth of the resin of 45 mm was obtained. Covington et al. used micro-stereolithography to create a microflu-idic structure for a biomimetic micro-sensor array-based olfactory device.41 Snowden et al. used microstereolithog-raphy to create 192 or 250 mm high

attachment, respectively.21,22 Soderberg et al. obtained minimum inhibitory concentration data for human wound infection-derived bacteria that were exposed to Zn+2.23 Different suscepti-bility rates were noted for Enterococ-cus sp., Proteus sp., Pseudomonas aeruginosa, (MICs of 8–32 mmol/L); Enterobacter sp., Escherichia coli, Klebsiella sp. (MICs of 4–8 mmol/L); Staphylococcus aureus, Streptococ-cus group B (MICs of 2–4 mmol/L); and Streptococcus groups A, C, and G (MICs of 0.5–2 mmol/L). Lansdown et al. also noted that topical application of zinc may increase migration of kera-tinocytes, breakdown of collagen, and removal of necrotic tissue.16 Although topical application of zinc oxide may lead to high (1,000–3,000 mmol/L) lo-cal zinc levels, these zinc levels may be associated with nontoxic and anti-oxidant properties.16,24–26 For example, Agren et al. showed that topical admin-istration of zinc oxide was associated with increased levels of zinc (1,540 µM) in wound fluid as well as lower rates of Staphylococcus aureus within wounds.26 Zinc oxide-containing paste bandages, known as Unna boots, are used to protect inflamed peri-ulcer skin in leg ulcers and reduce inflammation in lower extremity eczema with venous stasis; in these bandages, cotton gauze is saturated with zinc oxide paste. Elston suggested topical use of zinc compounds for acne treatment due to their anti-inflammatory and bacterio-static properties.15

Microneedles are miniaturized lancet-, thorn-, or hypodermic nee-dle-shaped devices that are used to penetrate the keratinized stratum cor-neum layer of the epidermis; this layer blocks transport of pharmacologic agents through the skin. Microneedles are sharp projections exhibiting at least one lateral dimension that is less than 500 mm.27,28 Microneedles are associ-ated with low levels of patient pain since these devices do not penetrate deep into the dermis, where Meissner’s corpuscles, Pacinian corpuscles, and large nerve endings are present.29–32 These devices facilitate transdermal delivery of pharmacologic agents, in-cluding protein-containing and nucleic acid-containing agents, by providing conduits through the stratum corne-

um layer of the skin.28 Donnelly et al. used an in vitro model to examine the movement of microorganisms through microneedle-fabricated pores.33 They showed that movement of Candida albicans, Pseudomonas aeruginosa and Staphylococcus epidermidis across Silescol® membranes was an order of magnitude lower for microneedle-fab-ricated pores than for 21G hypodermic needle-fabricated pores. On the other hand, adherence of microorganisms to microneedles was one order of magni-tude higher than adherence of micro-organisms to hypodermic needles. No microorganisms traversed viable por-cine epidermis that had been punctured with microneedles. Several investigators have recently considered the use of microneedles for treatment of skin conditions. For example, Doddaballapur described the use of microneedles for treatment of acne scars, stretch marks, scars, and wrinkles.34 Majid described a clinical study involving use of microneedles for treatment of atrophic facial scars. 94% of microneedle-treated patients ob-tained a reduction in scarring severity of one or two grades.35 More recently, Chandrashekar and Nandini described the combined use of microneedles and subcision for depressed acne scars.36 Lane described transdermal delivery of antimicrobial agents, including an-tibacterial and antifungal agents, by means of microneedles.37 He discussed systemic delivery of antimicrobial agents, including agents that cannot be orally administered (e.g., vancomycin), in a manner that exceeds the minimum inhibitory concentration or minimum microbicidal concentration. Further-more, the emergence of drug resistant microorganisms would be reduced and the course of therapy would be short-ened. In recent work, fabrication of mi-croneedles containing antimicrobial agents has been demonstrated. In one study, a master structure of a mi-croneedle array was produced by two photon polymerization.38 Microneedles were subsequently prepared out of an organically modified ceramic hybrid sol–gel (Ormocer®) material using a polydimethylsiloxane mold. These two photon polymerization/micromolding-fabricated microneedle arrays were

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Table I. Hole Fabrication Data

Input(µm)

Square Actual(µm)

% Holes Open

Input(µm)

Circle Actual(µm)

% Holes Open

800 656.25 ± 15.83 100% 800 No Open Holes 0%

700 429.2 ± 42.5 100% 700 No Open Holes 0%

600 245.9 ± 94.3 40% 600 No Open Holes 0%

550 245.5 20% 550 No Open Holes 0%

500 219.4 20% 500 No Open Holes 0%

450 199.6 20% 450 No Open Holes 0%

400 No Open Holes 0% 400 No Open Holes 0%

1 mm

0 2 4 6 8 10 12Line Number

Input FileActual Line

600

500

400

300

200

100

0

Line

Wid

th (m

m)

a

b

Figure 1. (a) Input line width and mea-sured line width dimensions for the line width resolution study. Error bars indicate standard deviation of mean values. (b) Image of diffusion-driven polymerization in a line width resolution test structure.

microfluidic components for electro-chemical flow detection; laminar flow conditions with volume flow rates of up to 64 mL min−1 were obtained with these structures.42 Stampfl et al. used microstereolithography to create struc-tures, including a free spinning turbine wheel and a fixed axe, out of hybrid sol–gels, hydrogels, and elastomers.43

They described processing of materi-als with a wide range of elastic moduli (0.1 MPa–8,000 MPa) by means of mi-crostereolithography. Neumeister et al. discussed fabrication of complex small-scale structures (e.g., small-scale micromechanical components) out of hybrid sol–gel materials.44 Choi et al. described use of a custom-built Digital Micromirror Device-based microste-reolithography system for fabrication

of microneedle arrays, microfans, and other high aspect ratio structures.45 Park et al. discussed fabrication of a 3×3 ar-ray of microcone cylinders by means of microstereolithography; a 5 mm wide tip and a maximum error of ~2 mm at the tip were obtained.46 More recently, Miller et al. used Digital Micromirror Device-based microstereolithography to create a hollow microneedle array out of an acrylate-based polymer.47

Carbon fiber electrodes were incorpo-rated within the microneedles; these electrodes were modified to facilitate monitoring of ascorbic acid and hydro-gen peroxide. Detection of hydrogen peroxide and ascorbic acid by the car-bon fibers within the integrated elec-trode-hollow microneedle devices was demonstrated. In addition, Park et al.

used a segment curing method to create micro-lens arrays; they also discussed the use of gray-scaled cross-sectional images to improve the surface profiles of microstereolithography-fabricated structures.48 Digital Micromirror De-vice-based microstereolithography has also been used to create prostheses and scaffolds for tissue engineering. For example, Gittard et al. utilized visible light dynamic mask microstereolithog-raphy to create carpal bone prostheses from patient computed tomography data.49 The stereolithography-fabricat-ed scaphoid and lunate prostheses ex-hibited ~50-mm thick layers. The min-imum compressive forces necessary for fracture of the scaphoid and lunate prostheses were 1360 N and 1248 N, respectively. Han et al. used a Digital Micromirror Device-based system to create three-dimensional scaffolds out of poly(ethylene glycol) diacrylate.50 These multilayered scaffolds were covalently conjugated with fibronec-tin to facilitate attachment of murine marrow-derived progenitor cells. Choi et al. described processing of porous biodegradable poly(propylene fuma-rate) scaffolds with ~100 mm inter-connected pores by means of Digital Micromirror Device-based microste-reolithography.51 More recently, Yasar et al. created branched structures for tissue engineering using Digital Mi-cromirror Device-based microstereo-lithography.52

Antimicrobial coatings were depos-ited on the visible light dynamic mask microstereolithography-fabricated mi-croneedles by means of pulsed laser deposition.53 In this physical vapor de-position process, material from a solid target is ablated using a high energy la-ser (e.g., an excimer laser). The atomic and molecular species generated by la-ser ablation exhibit an average kinetic energy of species of 100–1,000 kT.54,55 On the other hand, thermal deposition techniques provide species with ener-gies on the order of kT, which is 0.0259 eV at 300 K. Chemical reactions be-tween the substrate and the high energy species in the growing film serve to enhance film adhesion.56 In addition, thin films deposited by pulsed laser de-position exhibit high densities and low porosities. Pulsed laser deposition is well suited for film growth on polymer

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a

200 mm 200 mm 200 mm

b

c

d

200 mm 200 mm 200 mm

200 mm 200 mm 200 mm

200 mm 200 mm 200 mm

Figure 2. SEM images of eShell 200 microneedle array structures, which were produced using visible light dynamic mask microstereolithography. The geometries of the input STL file are (a) 750 mm x 250 mm base and 1,000 mm height for the “A” microneedle array structure, (b) 500 mm x 250 mm base and 1,000 mm height for the “B” microneedle array structure, (c) 750 mm x 250 mm base and 1,250 mm height for the “C” microneedle array structure, and (d) 500 mm x 250 mm base and 1,250 mm height for the “D” microneedle ar-ray structure. The images show the shorter axis of the microneedle, the longer axis of the microneedle, and a perspective view. The images were obtained at a 60º tilt.

substrates since deposition of many films can be performed at room tem-perature. In previous work, zinc oxide films were grown on silicon wafers us-ing pulsed laser deposition; CDC Bio-film Reactor and disk diffusion studies confirmed the antimicrobial properties of these materials.57

In this paper, the use of visible light dynamic mask microstereolithography combined with pulsed laser deposition to prepare antimicrobial microneedles was investigated. Studies were initially performed to determine the resolution of the microstereolithography system. Solid microneedles with four differ-ent geometries were produced; one of these geometries was subsequently se-lected for further evaluation. Needles in a cross shape, which may be used for wound closure, were also created using visible light dynamic mask microste-reolithography. Pulsed laser deposition was then used to coat microneedle ar-rays with silver or zinc oxide thin films. Scanning electron microscopy, atomic force microscopy, and profilometry were used to examine microneedle surface morphology, pulsed laser de-position-grown film structure, and film thickness, respectively. Penetration of the visible light dynamic mask micro-stereolithography-produced micronee-dles into porcine skin was confirmed by trypan blue staining. Agar diffusion assays were used to examine the inter-actions between coated microneedles and Escherichia coli, Staphylococcus epidermidis, and Staphylococcus au-reus bacteria. Our findings show that visible light dynamic mask microste-reolithography combined with pulsed laser deposition is an appropriate ap-proach for creating solid microneedle arrays with antimicrobial properties.

rEsults and discussion

Optical microscopy images of the test structures produced by means of visible light dynamic mask microste-reolithography are provided in Figure Bd–f. In these images, the largest input hole (800 µm) is on the left for the hole resolution images; the widest input line width (140 µm) is at the bottom for the line width images. As can be seen in these images, the lateral dimensions of the visible light dynamic mask micro-stereolithography-fabricated structures

are significantly larger than the lateral dimensions of the corresponding input files. The system was better at produc-ing holes with square inputs than those with circular inputs. It should be noted that the holes produced using square input file exhibit rounded corners. The square 800 µm and 700 µm input files were able to consistently produce holes; on the other hand, the corre-sponding circular input file produced no open holes. Although some holes were able to be produced for smaller input sizes, these holes were not con-sistently produced. The input values and measured values for the square and circular holes are provided in Table I. All of the holes were more than 140 µm smaller than the corresponding input dimensions; the hole size error ranged from 144–354 µm. As can be seen from the line width

resolution test image, only input struc-tures containing trenches with widths of 500 µm or greater produced trench-es in the corresponding test structures. A plot of input line width values and measured line width values across the largest gap (500 µm) for the line width resolution structure is provided in Fig-ure 1a. The measured line width values were determined by averaging the line widths of four different structures; the error bars indicate the standard devia-tion of the mean. The measured line width values were noted to be more than two times greater than the input line width values; the size discrepancy increased with the input line width. It is interesting to note that the 60 µm wide input line resulted in production of a line; this result indicates that a pixel does not need to be completely filled for the pixel to be illuminated.

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However, lines less than 60 µm wide did not result in illumination of pixels. This study indicates that visible light dynamic mask microstereolithography system software may completely po-lymerize material in locations where incomplete structures or surfaces exist in the corresponding input fi le. As an example, a feature with a size of one pixel, which lies between four pixels, may result in the illumination of four pixels; in this case, the measured di-mensions of the visible light dynamic mask microstereolithography system-fabricated feature would be much larg-er than the input dimensions. Scanning electron micrographs of the uncoated four different geometries are shown in Figure 2. The images show the shorter axis of the micronee-dle, the longer axis of the microneedle, and a perspective view; all of these images were obtained at a 60º tilt. The input and measured dimensions of the microneedles are provided in Table A. For all of the microneedle geometries, the measured lateral dimensions were larger than the input lateral dimen-sions. In contrast, the measured vertical dimensions were shorter than the input vertical dimensions. Although the in-put heights were identical, the 750 µm

wide structures were taller than the 500 µm wide structures. As shown in Table A, the lateral dimensions of the microneedle were larger than the corresponding input data. The heights of the microneedles were shorter than the corresponding input data. In visible light dynamic mask microstereolithography, the sur-face of the STL model is converted in a process that is known as tessellation to a series of polygons.65 This series of polygons is sliced into cross-sectional layers, which are subsequently used for fabrication of the structure (e.g., the microneedle device) in a layer-by-layer manner. Variations between the input dimensions and measured dimen-sions are ascribed to translation of the STL model to the physical structure. The discrepancy in lateral dimensions, particularly the fact that the measured dimensions are larger than the input di-mensions, can be attributed to the fact that the lateral voxel size (155 µm) is larger than the pixel size (70 µm). There are also discrepancies in the vertical dimensions between the test structures and the visible light dynamic mask mi-crostereolithography-fabricated struc-tures. The pixel size in the projection mask corresponds to approximately 70

µm; features below a certain size may not result in production of features by the visible light dynamic mask micro-stereolithography system. Higher magnifi cation top-down and side views were used to obtain z-step size, tip radius of curvature, and as-pect ratio measurements (Figure 3). The build layers can clearly be seen in the scanning electron micrographs of the visible light dynamic mask microstereolithography-produced mi-croneedles. The z-direction step size was measured to be 29.89 ± 0.61 µm. The radius of curvature was measured to be 18 µm. The aspect ratio of wide face of the D geometry microneedle was obtained by dividing the average z-step size by twice the x and y step distance from layer to layer (via the top-down image) over the entire mi-croneedle length. The average aspect ratio measured in a layer-by-layer man-ner along the entire length of the needle was 2.4 ± 0.3 to 1; the net aspect ratio (total height divided by width) was de-termined to be 2.2 to 1. Other factors that contribute to dis-crepancies between input and mea-sured dimensions in visible light dy-namic mask microstereolithography are diffraction, refraction, and pho-toinitiator diffusion.61–63 Quantitative analysis of voxel size spreading due to diffraction has been performed by Sun et al.61 In the visible light dynamic mask microstereolithography system, the halogen bulb used as a light source emits a spectrum that contains a range of wavelengths; in addition, the photo-initiator can be excited by a range of wavelengths. Light of various wave-lengths will refract in different amounts as it passes through the material. In addition, already polymerized mate-rial will refract differently than unpo-lymerized material.62 Diffusion-driven polymerization can result in fusing of nearby features. In diffusion driven polymerization, electronically-excited photoinitiator molecules diffuse out of the voxel and produce membrane-like features between nearby features. The membrane-like features of diffusion driven polymerization may be clearly observed in some of the line resolu-tion structures between the larger lines (Figure 1b). This phenomenon has been observed in other photopolymer-

200 mm

100 mm

Radius of curvature = 18 mm

Figure 3. SEM images of the “D” mi-croneedle array structure; the input di-mensions for this structure are (a) 250 × 500 µm base and (b) 1,250 µm height. Plan view and 90º tilt images are pre-sented; the radius of curvature of the tip is shown on the 90º tilt image.

a1 mm

b

Figure 4. (a) SEM image of micronee-dles on a cross-shaped substrate that was produced by visible light dynamic mask micro-stereolithography. (b) CAD drawing of the input fi le that was used to produce the structure using visible light dynamic mask micro-stereolithography.

a

b

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ization processes, including two pho-ton polymerization.63 The appearance of layers in the polymerized structures indicates that the voxel height is larg-er than the step size and that there is signifi cant overlap between the voxels during processing. The visible light dynamic mask mic-rostereolithography system was able to produce 32 microneedle arrays in a sin-gle, four-hour batch when using a 30-µm layer spacing. Both the low input material cost and the high production rate (approximately 7.5 minutes per microneedle device) make the visible light dynamic mask microstereolithog-raphy system an appealing approach for scalable production of micronee-dles. A microneedle device with a cross-shaped geometry was also able to be produced with visible light dynamic mask microstereolithography; the STL fi le used to make this device is shown in Figure 4a. A scanning electron mi-crograph containing two arms of the cross-shaped microneedle device is shown in Figure 4b. A device with this cross-shaped geometry may be used for facilitating wound healing and holding the skin together. Porcine skin and trypan blue have been used to as-sess the functionality of microneedles for transdermal drug delivery.47,64,65

Figure 5a and b shows optical micro-graphs of microneedles before inser-tion into skin and after insertion into skin, respectively, which show that the microneedles remain intact after enter-ing skin. Optical micrographs showing delivery of trypan blue into micronee-dle-fabricated pores within skin are shown in Figure 5c and d. The blue colored spots (identifi ed by arrows) indicate penetration of trypan blue dye through the stratum corneum layer by the microneedle as well as localization of trypan blue inside the microneedle-generated pore. The pores are larger in one direction than the other, which is in accordance with the microneedle di-mensions. Optical microscopy images of the pulsed laser deposition-coated mi-croneedle devices are shown in Figure 6a and b. The silver-coated micronee-dle device (Figure 6c), zinc oxide-coated microneedle device (Figure 6b), and uncoated microneedle device (Fig-

ure 6a) are shown. Differences in the surface roughness of the zinc oxide-coated surface and the silver-coated surface are noted in Figure 6e and f. Atomic force micrographs obtained in tapping mode of the zinc oxide and sil-ver thin fi lms on silicon substrates are shown in Figure 7. Root mean squared surface roughness values for silver thin fi lms and zinc oxide thin fi lms were 1.4 nm and 1.3 nm, respectively. The low roughness values of the thin fi lms indicate that splashing was not signifi -cant.51 The thicknesses of the coatings were measured by profi lometry to be 53.5 ± 8.8 nm for Ag and 287.2 ± 30.3 nm for ZnO. Deposition rates were cal-culated from profi lometry data; rates of 0.018 nm/pulse and 0.10 nm/pulse were determined for silver and zinc ox-ide, respectively. In comparison, Choi et al. observed a zinc oxide growth rate of 0.017–0.033 nm/pulse for pulsed laser deposition involving an ArF ex-

cimer laser.66 Warrender et al. observed a silver growth rate of 0.0018–0.006 nm/pulse for pulsed laser deposition involving a 248 nm KrF excimer.67 Variations in deposition rate can be attributed to laser fl uence, target tem-perature, pressure, and other param-eters.54,66,67

Images of the agar diffusion assay results for silver-coated microneedle devices, zinc oxide-coated micronee-dle devices, and uncoated microneedle devices are presented in Figure 8. The agar diffusion assay results indicate that silver thin fi lms and zinc oxide thin fi lms provided the microneedle devices with antibacterial properties. Both thin fi lms had signifi cant effects against Staphylococcus epidermidis and Staphylococcus aureus. On the other hand, signifi cant effects against Escherichia coli were not noted. The silver-coated microneedle devices produced zone voids of growth; in

1 mm 1 mm

1 mm 1 mm

Figure 5. (a) Optical mi-crograph showing two mi-croneedles before insertion into porcine skin. (b) Optical micrograph showing two microneedles after inser-tion into porcine skin. (c,d) Optical micrographs show-ing delivery of trypan blue into microneedle-fabricated pores within porcine skin.

a b c

d e f

2 mm

50 mm50 mm50 mm

Figure 6. (a–c) Optical microscopy images and (d–f) high magnifi cation SEM images of (a,d) uncoated, (b,e) zinc oxide-coated, and (c,f) silver-coated microneedle array structures.

a b

c d

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contrast, the zinc oxide-coated mi-croneedle devices produced zones of inhibited or reduced growth. Some of the thin films remained on both the sil-ver-coated microneedle devices and the zinc oxide-coated microneedle devices after 24 hours of contact with the agar. In comparison, the uncoated arrays had no antimicrobial effect. As mentioned in the introduction, zinc oxide exhib-its weaker bacteriostatic inhibition of Gram-negative bacteria than of Gram-positive bacteria.68 The agar diffusion assay confirmed previous findings that pulsed laser deposition-grown thin films of both silver and zinc oxide pro-vide antibacterial properties.38,57

conclusions

In this study, we have demonstrated that visible light dynamic mask mi-crostereolithography is an appropri-ate technique for scalable production of microneedle devices. Although the visible light dynamic mask microste-

reolithography system reproduces lat-eral dimensions with good fidelity, the lengths of the tapering structures were significantly reduced. The successful fabrication of microneedles on cross-shaped substrates, which may be for holding the skin together and facilitat-ing wound healing, as well as square substrates demonstrates that visible light dynamic mask microstereolithog-raphy provides flexibility for creating medical devices with complex shapes. The combination of dynamic mask stereolithography with pulsed laser de-position was used to prepare acrylate-based polymer microneedle arrays with antimicrobial properties; these devices may be useful for wound treatment. Agar diffusion assays confirmed that pulsed laser deposition-grown silver and zinc oxide coatings exhibit anti-microbial activity. The ability to pro-duce microneedle arrays for treatment of skin infections by means of visible light dynamic mask microstereolithog-

raphy and pulsed laser deposition is a promising step towards bringing mi-croneedle technology into wider use.

acknowlEdgEmEnts

Portions of the research in this pa-per were funded from Office of Naval Research grant N00014-07-1-1099.

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Figure 8. Agar diffusion assay results for un-coated (left), Ag-coated (center), and ZnO-coated (right) “D” geometry mi-croneedle array struc-tures. The microneedle array structures were evaluated using Esch-erichia coli (top), Staphy-lococcus epidermidis (center), and Staphylo-coccus aureus (bottom). The microneedle array structures examined in this study were distrib-uted over 18 mm ×18 mm areas.

Control Ag ZnO

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S. aureus

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Shaun D. Gittard, Philip R. Miller, Chunming Jin, Timothy N. Martin, Ryan D. Boehm, Nancy A. Mon-teiro-Riviere, and Roger J. Narayan are with the Joint Department of Biomedical Engineering, Uni-versity of North Carolina and North Carolina State University, Chapel Hill, NC 27599; Monteiro-Riviere is also with the Center for Chemical Toxicology Research and Pharmacokinetics, Department of Clinical Sciences, North Carolina State University, Raleigh, NC 27695, USA; Bret J. Chisholm, Shane J. Stafslien, Justin W. Daniels, and Nicholas Cilz are with the Center for Nanoscale Science and Engineering, North Dakota State University, 1805 Research Park Drive, Fargo, ND, 58102; and Ad-nan Nasir is with the Department of Dermatology, University of North Carolina, Chapel Hill, NC, and Wake Research Associates, 3100 Duraleigh Rd. Ste. 304, Raleigh. Dr. Narayan can be reached at (919) 696-8488, e-mail roger_narayan@unc.edu.

Roger Narayan is a TMS Member!

To read more about him, turn to page 9. To join TMS, visit www.tms.org/Society/Membership.aspx.