Chitin Microneedles for an Easy‐to‐Use Tuberculosis Skin Test

• DOI: 10.1002/adhm.201300185
• Publication Date: 2013-08-26T09:22:42Z
• Authors: Jungho Jin; Valerie Reese; Rhea Coler; Darrick Carter; Marco Rolandi
• Keywords: Skin test

An easy-to-use tuberculosis skin test is developed with chitin microneedles that deliver purified protein derivative at the correct depth and result in a positive BCG-immunized guinea pigs Microneedles combine transdermal delivery capabilities of hypodermic needles advantages dermal patches.1-4 Upon insertion, create small holes stratum corneum, outer most layer, to afford improved drug diffusion into dermis, similarly needles. At same time, application safe painless like patches.1 intradermally therapeutics vaccines. Examples include insulin,5, 6 hormones,7, 8 DNA,9, 10 Bacillus Calmette-Guérin (BCG),11 influenza.12 Intradermal used diagnostic tests for allergies (TB). This intradermal difficult achieve requires trained personnel.13 Coated are ideally suited precise diagnostics. To best our knowledge, not such example currently exists. Here, we develop an array made chitin, natural polysaccharide, demonstrate proof-of-concept TB diagnostics (Figure 1). silicon,1, 14, 15 metals,1, 16, 17 synthetic polymers,1, 18, 19 and, recently, biomaterials as silk20 chitosan.21 Silicon metal offer high elastic modulus tensile strength ensure penetration. These usually coated substance needs be delivered, or fabricated opening mimics traditional needle. Polymer via replica molding rigid microneedle master lower manufacturing costs. Dissolvable biodegradable polymer time-delayed controlled-release vaccines therapeutics.1 Polymers required successful insertion skin.22 Among these polymers, materials particularly desirable. Chitin [poly(β-(1,4)-N-acetyl-d-glucosamine] naturally abundant which mechanically robust, nontoxic, physiologically inert, biodegradable.23, 24 We have recently demonstrated micro- nanostructures easily microcontact printing.25, 26 Most lengths allow rather than subcutaneous intramuscular commonly achieved regular can enhance protective immune responses because dendritic cells other effectors reside close surface skin.27, 28 Skin antigens good markers allergy29, 30 tests.13 affects one-third world's population.31 Although treated prevented aid antibiotics BCG, diagnosis still prime importance infections latent asymptomatic. The common Mantoux 1b), administered every year millions people United States alone including healthcare professionals teachers. perform test, dose "PPD"—containing mixture antigens—is injected subject's forearm using needle.32, 33 For needle must inserted 5–15° angle within layer (≈1 mm below surface). Insertion wrong results too shallow deep PPD failed 1c,d). Devices based on miniaturized been developed.34, 35 Additionally, multiple arrays spring-activated mini-needles excess (Heaf gun Sterneedle gun) utilized provide user-friendly higher sensitivity.36-38 ideal means administering designed precisely controlled by device geometry user.39 Despite being diagnostics, date intensively explored vaccines, but testing vivo model pigs. this first demonstration more conventional vaccine purpose. from aluminum master20 polydimethylsiloxane (PDMS) mold 2). process, use 2.0% chitin/hexafluoroisopropanol (HFIP) gel 2a). obtain gel, sponge lyophilization 0.5% chitin/HFIP solution then redissolve HFIP. porous less crystalline commercially available β-chitin powder has solubility HFIP S1, Supporting Information). Microneedle fabrication most-concentrated solutions obtained simple mixing (0.5% w/v)26 yields defective weak microneedles. During evaporation only little left due solvent content, resulting very thin sidewalls. poured onto PDMS drying performed centrifuge 2b). In fashion, pushed during process ensures quality microneedles.21, 40 Without centrifugation, capillary stresses41 cause uncontrolled shrinkage delaminate mold, poorly replicated structures S2, Using centrifugation uniform 750 μm-tall (300 μm-wide base) 3). tip diameter ≈30 μm. sharper original Al 3a,b). likely solvent-free poor hydrogen bond acceptor highly volatile, all evaporates process.26 confirm absence HFIP, tissue engineering substrates show no cytotoxicity,42-44 observe signs inflammatory reaction control samples. transparent densely entangled nanofiber morphology expected self-assembly S3, Information).25, Self-assembled films 2.5 GPa exceeding 120 MPa.45 robust. From compression measure buckling force mechanical failure single ≈0.2 ≈0.5 N, respectively S4, Information).40, 46 exceeds (<0.1 N)22 reasonable safety factor. penetration human cadaver 3c,d) further verified additional silicone rubber mimic pig sample S5, dimensional stability retained even after one storage under ambient condition, may remain intact unless exposed hydrolytic enzymatic environment, found air. proof-of-concept, 4). developing they respond delayed-type hypersensitivity (DTH) similar seen humans, do need kept BSL-3 environment.47, 48 Arrays six per varying amounts concentrated (1 μg μL−1) solution: 0 (control), 40, 60, 80 μL. samples designated PPDX, where X amount coating Coating occurs immersing conditions. stable agent it stored least 1 37 °C49, 50 several days months extreme conditions (100 °C, −28 °C) without any deterioration its biological activity.50, 51 potency when dried followed redissolution.52 insoluble water-based significant swelling degradation properties, would compromise their ability penetrate skin. promising feature compared ones biocompatible chitin's aqueous environment affords efficient mostly water medium.1 Furthermore, physiological inertness enables inducing DTH 24–72 h. All mounted adhesive patches prior 4e). applied back shaved comparison, needle-based saline standard PPD—5 unit (5-TU), 100 μL—in animals. As diagnostics,13 size erythema eventual induration measured h time points, 4a,b). expected, both readouts 4a,b,f). does appreciable response reading test. advantage dissolve readily upon skin, reduces risk potential material itself. Long-term biodegradability enzymes event accidental breakage skin.53, 54 PPD40 4a,b,g). Assuming 100% efficiency, 40-μL should correspond ≈800 TU PPD, well current dosage However, current-coating strategy unlike delivered solid. It possible solid form effective dose. fact, Heaf dip-coated contains 000 mL−1. light, negative μL surprising. h, PPD60 PPD80 clear 5.5 6.5 mm, comparable observed animals protocol 4c,d). grows 6.4 induration, indication On hand, transforms 4.9 4b,i). 5-TU injection ≈8 4d). measurable considered S6, confirms rule out false positives, inject saline-sensitized concentration S7, convenient tool, although systematic follow-up studies addressed optimize loading efficiency.11 Nevertheless, approach produces give true-positive outcome. conclusion, centrifugation-assisted gel. strong N 0.1 puncture what advantageous length user. such, expect able microneedle-based easier perform, painful, rate administration errors. Finally, opens doors infectious diseases practice. Materials: squid extract (Industrial Research Ltd., New Zealand) hexafluoro-2-propanol (Oakwood Products, Inc.) received. (Sylgard 184 kit) cast mold. (Tubersol, Sanofi Pasteur Inc., USA) received Silicone scar-care sheets (ScarAway) Preparation PPD-Coated Patches: cut each dispensed vial separately, immersed vial. basic 0.05–0.08 content (TU), typically 5–250 used. Numerically, treat (not ultimately coats microneedles) corresponds 800, 1200, 1600 TU, respectively. room temperature fume hood 2 d. After completely dried, patch. Vivo Test Guinea Pigs: immunized 5 × 104 CFU BCG Connaught (TheraCys, Live (Intravesical), Pasteur, testing. DTH, Tubersol given intradermally. backs sufficient finger pressure. Patches overnight removed next morning. Cohesive flexible bandage wraps (Fisher Healthcare, order protect checked respectively, measuring mean diameters and/or photographs taken accordingly. Infectious Disease Institute (IDRI) established policies ethical sensitive care research approval Office Laboratory Animal Welfare (OLAW) National Institutes Health (NIH). IDRI animal protocols reviewed approved IACUC. M.R. acknowledges funding Coulter Foundation, University Washington Center Commercialization, 3M Nontenured faculty award, Institute. work was supported part Allergy Diseases, (Grants AI044373 AI078054) Contract HHSN272200800045C. authors acknowledge Chase Ruebel (UW), Daniel Hutchins Susan Baldwin assistance some experiments. service readers, journal provides supporting information supplied authors. Such peer re-organized online delivery, copy-edited typeset. Technical support issues arising (other missing files) Please note: publisher responsible functionality Any queries content) directed corresponding author article.