Chitin Microneedles for an Easy‐to‐Use Tuberculosis Skin Test
Authors: Jungho Jin, Valerie Reese, Rhea Coler, Darrick Carter, Marco Rolandi
Journal: Advanced Healthcare Materials (2013)

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