Silk Fibroin Microneedles for Transdermal Vaccine ... - ACS Publications

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Silk fibroin microneedles for transdermal vaccine delivery Jordan A. Stinson, Waseem K. Raja, Sangun Lee, Hyeun Bum Kim, Izzuddin Diwan, Stephen Tutunjian, Bruce Panilaitis, Fiorenzo G Omenetto, Saul Tzipori, and David L Kaplan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00515 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on December 31, 2016

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TITLE Silk fibroin microneedles for transdermal vaccine delivery AUTHORS Jordan A. Stinson1, Waseem K. Raja1,2, Sangun Lee3, Hyeun Bum Kim4, Izzuddin Diwan1, Stephen Tutunjian1, Bruce Panilaitis1, Fiorenzo G. Omenetto1, Saul Tzipori3, and David L. Kaplan1* AUTHORS ADDRESS 1 Department of Biomedical Engineering, 4 Colby Street, Tufts University, Medford MA 02155 2 Department of Brain and Cognitive Sciences, 43 Vassar Street, Massachusetts Institute of Technology, Cambridge MA 02139 3 Department of Infectious Disease and Global Health, 200 Westboro Road, Tufts University Cummings School of Veterinary Medicine, North Grafton MA 01536 4 Department of Animal Resources Science, Dankook University, Cheonan 31116, South Korea *CORRESPONDING AUTHOR David L. Kaplan Chair, Department of Biomedical Engineering, Tufts University, Medford, Massachusetts [email protected] ABSTRACT Microneedles represent an exciting departure from the existing parenteral injection paradigm for drug delivery, particularly for the administration of vaccines. While the benefit of delivering vaccine antigens to immunocompetent tissue in the skin has been established, there have been varying degrees of success using microneedles to do so. Here, we investigate the use of silk fibroin protein to produce microneedles and evaluate their ability to deliver vaccines against influenza, Clostridium difficile, and Shigella. Fibroin protein from the silkworm Bombyx mori possesses suitable properties for use in a microneedle system, including all-aqueous processing, mechanical strength in dried formats, biocompatibility, and the ability to temperature stabilize biomacromolecules. As such, this biomaterial combines the processing and biocompatibility advantages of a dissolving microneedle system with the product stability and mechanical strength of coated insoluble microneedles. Through successful vaccination of mice against three separate antigens, we establish that silk fibroin is well-suited for use as a solid-coated microneedle delivery system and offers long-term potential similar to the leading microneedle biomaterials. KEYWORDS Influenza, Clostridium difficile, Shigella, Device Fabrication, Soft Lithography

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INTRODUCTION Developing nations face a significant public health challenge in the form of improving vaccination coverage. It was recently estimated that of 129 million children born in 72 different developing countries, only 66 percent received a hepatitis B vaccine, 29% received a Haemophilus influenzae type B vaccine, 8% received a rotavirus vaccine, and 7% received a meningococcal vaccine1. Unfortunately, the access to vaccines in these nations is limited by financial costs and distribution logistics. Furthermore, the activity and efficacy of vaccines are sensitive to temperature and demand refrigeration throughout the distribution and transportation processes2. These limitations have driven the investigation of alternative delivery routes enabled through vaccine delivery devices that are simple, cheap to manufacture, and thermostable. Transdermal devices, intended for delivery of the vaccine payload through the skin, meet some of these important criteria. As the body’s first barrier to external pathogens, the skin is a unique target for vaccines as it is an immunocompetent tissue with populations of Langerhans cells in the epidermis and dendritic cells in the dermis3-4. These cells will uptake, process, and present vaccine antigens to naïve B- and Tcells, leading to efficient and robust immune responses5-6. One such transdermal vaccination approach is through the use of microneedle devices, which are patches with arrays of needles less than a millimeter in height. These devices offer the potential to stabilize vaccines in the solid phase and be self-administered, removing the barriers of cold storage distribution and access to a physician which can limit vaccine coverage in remote locations of the world7. These devices also present an advantage over existing parenteral administration methods by reducing the pain and anxiety associated with hypodermic injections. The benefits of microneedle administration have been modeled with influenza vaccine and were estimated to increase patient intent-to-vaccinate by greater than 20%, potentially saving up to $2.6 billion during a flu season8. The cost per vaccination could be further reduced through the use of microneedle patches, as intradermal delivery has led to robust immune responses with lower doses of antigen9. Here, we present our work to assess the ability to use microneedles prepared from a unique biomaterial, silk fibroin, for the delivery of vaccines against influenza, Clostridium difficile, and Shigella. Prepared from the silkworm Bombyx mori, silk fibroin protein offers desirable material characteristics for use in such a microneedle delivery system10. Similar to dissolving biomaterials previously used in microneedles (such as carboxymethylcellulose11, PLGA12-14, PVA15-16, hyaluronic acid17-18, and maltose19-20), silk can be processed into microneedles under all-aqueous conditions without the need for ultraviolet crosslinking, elevated curing temperature, or high molding pressure21-22. As a copolymer with hydrophobic blocks connected by hydrophilic segments, silk fibroin can be post-processed to increase crystalline β-sheet regions, eliminating any susceptibility to humidity-induced degradation which can be a storage problem for some sugar-based microneedles23-25. Silk fibroin is also a resilient and mechanically strong material, while remaining biocompatible and degrading into non-inflammatory products in vivo26-30. In comparison to other materials used to fabricate microneedles, fibroin has also been demonstrated to protect certain biomacromolecules during exposure to elevated temperatures31-34. With these material properties, we and others have previously developed methods to produce silk fibroin microneedles and have studied the delivery of model therapeutics with them21-22, 35-37. In the current work, we build upon these prior studies to investigate the efficacy of using a vaccine-coated silk microneedle to deliver both commercial and research-stage protein antigens to evaluate the potential of this biomaterial in a transdermal delivery application.


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MATERIALS AND METHODS Silk Fibroin Solution Preparation Silk fibroin protein solution was prepared from Bombyx mori cocoons according to the standard methods as previously described in the literature38. B. mori cocoons were cut open to remove the silkworm and were subsequently diced into pieces roughly 3-4 square centimeters in size. Five grams of prepared cocoons were extracted for 30 min in an aqueous alkaline solution of 0.02 M sodium carbonate (SigmaAldrich, St. Louis, MO) to remove the gum-like silk sericin protein. Following a deionized (DI) water rinse process, the silk fibroin was allowed to dry overnight in a fume hood before being dissolved in a 9.3 M lithium bromide solution (Sigma-Aldrich, St. Louis, MO) for 4 h at 60°C. This dissolved silk solution was then transferred to Spectra/Por dialysis membrane tubing (3,500 Da, SpectrumLabs) for dialysis for two days, with six periodic water changes performed. Resulting solution (~5–6 w/v%) was concentrated overnight on the bench to achieve a final concentration of 7–8 w/v%. This solution was centrifuged two times for 20 min at 8,600 rpm before storage at 4°C to remove any residual particulate matter. Final concentration was determined by weighing the residual silk mass after drying a known volume of solution. Fabrication of Silk Microneedle Arrays The fabrication of polymer-based microneedles is well-described in literature and consists of steps to produce molds into which polymer is cast and dried to form microneedle structures7,12-13. Negative poly(dimethyl siloxane) (PDMS) molds (Sylgard 184, Dow Corning, Midland, MI) for silk microneedle fabrication used in these studies were prepared from existing master positive molds whose fabrication has been previously described22. Briefly, computer numerical control machining was used to drill a chamber in a wax block that is subsequently milled with drill bits to create a template array of microneedles. A profile tool (Bits and Bits, Silverton, OR) was then pressed into each machined needle well to remove any surface irregularities and create the final microneedle geometry. Epoxy (Araldite 502, Electron Microscopy Sciences, Hatfield, PA) was cast into the machined wax mold and cured for 48 h at 80°C. This master positive was inspected through SEM prior to further soft lithography steps to generate identical daughter molds. PDMS was then cast around the master epoxy positive mold to form the negative mold from which silk microneedles were produced. The final PDMS molds were patterned with a 20 x 20 array of conical microneedles, each with the geometry of 700 µm height, 15 µm tip diameter, and 360 µm base diameter. The final microneedle array had an area of 1.5 cm by 1.5 cm. 800 µL of prepared silk fibroin solution (~6–8 w/v%) was pipetted into the PDMS molds and centrifuged at a rate of 5,000 rpm for 20 min at 4°C to force the solution into the needle cavities and evacuate any trapped air. After centrifugation, any remaining bubbles were removed with a pipette, and the mold trenches were completely filled with silk solution (total volume ~3.5 mL). The filled molds were left under ambient temperature and humidity conditions for 2–3 days to fully dry. The dried silk microneedle devices were carefully removed from the molds and placed in a vacuum chamber to water-vapor anneal for 24 h at ambient temperature24. After annealing, the edges of the patches were manually trimmed and the patches were pinned flat onto a PDMS surface prior to antigen coating. Silk Microneedle Antigen Coating Process Vaccine coating of the silk microneedle devices was performed in a two-step process to pre-wet the needle surfaces and then evenly distribute the final therapeutic formulation across the device. Although the antigen can be formulated into the bulk material of the silk devices, faster release kinetics and better immune responses have been observed from coated formulations on the silk-based devices21. To pre-wet the microneedles, 100 µL of deionized water is carefully pipetted into multiple (5-10) droplets on the surface of the microneedle tips. This volume was determined as adequate to spread to cover the full surface of the array without spilling over the edges of the patch. These droplets are left on the microneedles and monitored until they have combined to hydrate the silk material surface (~5 minutes).



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75 µL of the deionized water is then carefully pipetted off the patch prior to the addition of up to 100 µL of antigen-containing coating solution to the pre-wet needle surface. This process allows the dosage to be controlled through the volume/concentration of vaccine added to the patch, with no waste due to spilling off of the patch. This coating is dried overnight at room temperature to deposit a uniform layer of antigen (additional information in SI). All antigens used in these studies were formulated with 1% (w/v) silk fibroin (60 min extraction) to impart thermal stability during drying and storage but not to inhibit antigen release from the microneedle coating. For some studies, adjuvant was also included in the coating formulation to assess compatibility of the adjuvant with silk, safety during intradermal administration, and the effect on overall immune response to the protein antigen. Commercially-available vaccine was formulated to include silk fibroin but was not modified in any other manner. Antigen Selection and In Vivo Vaccination For this study, we aimed to assess the vaccination performance of the silk microneedles against three pathogens: influenza, C. difficile, and Shigella. Details for each antigen, including administration schedule and study design are included below. Influenza Influenza is one of the most well-studied vaccine targets for microneedle systems as its annual administration would be improved by a simpler administration technique that produces the same efficacy as subcutaneous injection. This target has been successfully delivered into murine skin using other microneedle materials and device formats, with ongoing clinical trials and an FDA-approved product (Fluzone® ID, Sanofi-Pasteur, Swiftwater, PA)18, 39-43. Quadrivalent influenza vaccine (Fluzone® 20142015, Sanofi-Pasteur, Swiftwater, PA) was kindly provided by our collaborator Dr. Patricia Hibberd at Harvard Medical School for the purpose of investigating transdermal dose response using silk microneedle delivery. Previous studies have suggested that 6 µg of influenza antigen can produce a strong immune response in BALB/c mice while a half-dose (7.5 µg per strain) was immunogenic in humans when delivered transdermally18, 41, 43. With the silk microneedle system, we sought to determine the strength of immune response to doses spanning this antigen range, including 12 µg, 6 µg, and 1 µg per patch (n =5). The immunization schedule consisted of a primary immunization with a single boost after two weeks to more quickly induce an IgG secondary immune response. Submandibular serum samples were collected from mice prior to each immunization and 2 weeks after the final administration event. Clostridium difficile With recent estimations suggesting nearly 500,000 C. difficile infections occurred in 2011 in the United States alone, there is a significant clinical need for a prophylactic vaccine to protect individuals against this pathogen44. As transdermal delivery has been shown to produce both local and systemic immune responses, microneedle-based delivery of C. difficile vaccine antigens may provide advantages over other routes of administration4-5. Chimeric toxin A/toxin B (cTxAB) vaccine was prepared and coated onto silk microneedles at a dose of 10 µg with 1 µg of the adjuvant dmLT45-46. This adjuvant, dmLT, is nontoxic double mutant Escherichia coli heat-labile toxin [LT(R192G/L211A)] and has been considered a good mucosal adjuvant. We selected dmLT for this study as the mucosal immune responses, including IgA, induced by vaccination are important to protect the host against C. difficile infection. This preparation was compared to an intradermal (ID) injection with the same composition, as well as a control formulation from previous study of 10 µg of cTxAB vaccine in PBS with alum adjuvant injected intraperitoneally (IP)45. All mice (n = 5 per group) were immunized three times at 2-week intervals, followed by a challenge with 106 spores of the toxigenic UK6 strain of C. difficile. Serum samples from all animals were collected 1 day prior to each immunization. Shigella Similar to C. difficile, Shigella bacteria cause severe diarrhea that can be potentially fatal, particularly in children less than five years old. Recent estimates suggest that there were over 88 million Shigella


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infections in Africa and South Asia in 2013 alone47. As shigellosis most commonly occurs in developing regions of the world with limited access to clean water, a vaccination strategy against these bacteria would be most effective if it was amenable to a door-to-door campaign. Microneedles provide the simplicity for such an immunization campaign, while also providing the humoral immunity believed to be necessary for protection against multiple strains of Shigella bacteria at mucosal surfaces48. For this study, fusion protein of the Shigella antigens IpaD and IpaB was prepared as these antigens are conserved across Shigella strains and known as protective antigens in a mouse model49. Microneedles were prepared with 30 µg of IpaDB fusion protein and 1 µg of dmLT adjuvant and compared to 10 µg of IpaDB protein and 1 µg of dmLT injected intradermally and intramuscularly. As with C. difficile protection, protection against shigellosis is mediated by mucosal immune responses so the dmLT adjuvant was selected for this study as well. A larger dose was coated onto the microneedles to account for the expected incomplete delivery of antigen (~1/3 efficiency) that had been observed in other studies. Mice were immunized on day 0, 14, and 28 and their responses were tracked biweekly through serum IgG levels plus vaginal, saliva, and fecal IgA levels. Microneedle Administration All animal studies were approved by the Institutional Animal Care and Use Committee of Tufts University Division of Laboratory Animal Medicine. Female BALB/c mice (Charles River Labs, Wilmington, MA) were obtained for the in vivo vaccination experiments with influenza (8-10 weeks old) and Shigella (4-6 weeks old). For the C. difficile vaccination experiments, female C57BL/6 mice (4-6 weeks old, Charles River Labs, Wilmington, MA) were used. Pre-immunized blood samples were obtained via submandibular bleeds and were collected in serum separator tubes (Amber Microtainer 365978, Becton Dickinson, Franklin Lakes, NJ). Serum was collected from the tubes following centrifugation at 13,400 rpm for 5 min and was stored at -20°C until analysis. Sample volumes were limited by IACUC protocol to a total of 140-180 µL every two weeks. To administer microneedle patches, animals were anesthetized with and maintained under isofluorane while the upper region of the back was shaved and treated with a depilatory cream (Nair™ Sensitive Formula, Church and Dwight). The silk microneedle device was administered by pinching the patch and skin between the index finger and thumb and then covering it with a piece of adhesive dressing (Tegaderm™, 3M, St. Paul, MN; image in SI). To ensure complete antigen release from the exposed coating surfaces in the dermis, microneedles were administered for 24 h before removal. Injection controls and experimental conditions were administered intradermally, subcutaneously in the dorsal region between the shoulders, intraperitoneally, and/or intramuscularly in a left hind leg for each antigen target. Microneedles were examined for integrity before and after microneedle administration in vivo (image in SI). Influenza Vaccine Transdermal Immunogenicity To evaluate the success of silk microneedle vaccine delivery, the level of serum IgG antibodies against the vaccine influenza strains was determined through indirect ELISA. Microtiter plates (Nunc Maxisorp 96 well, Thermo Fisher Scientific, Waltham, MA) were coated with 100 µL of influenza vaccine antigen at a concentration of 1 µg/mL in carbonate buffer (0.05 M, pH 9.5) and stored overnight at 4°C. Wash buffer (1X PBS with 0.01% Tween-20) was prepared and used to wash the wells of the microtiter plate three times before 200 µL of blocking solution (1% BSA in wash buffer) was added and incubated for 2 h at 25°C. Serial dilutions of the animal serum samples were prepared and 100 µL of these dilutions was added to triplicate wells after three plate washes. Sera was incubated for 2 h at 25°C in the plates to promote binding. Another wash step was performed before 100 µL of secondary antibody (biotinylated goat anti-mouse whole IgG, Sigma-Aldrich, St. Louis, MO) was added and incubated for 1 hour at 25°C. A final wash step was performed prior to developing the assay through the addition of 3,3’,5,5’tetramethylbenzidine (TMB, Sigma-Aldrich, St. Louis, MO). The reaction was stopped with sulfuric acid after 5-10 min of development and the absorbance of the samples was read at a wavelength of 450 nm. A four-parameter logistic curve fit was applied to the absorbance-dilution curves for each serum sample and



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was used to determine endpoint titer, defined as the dilution at which the absorbance signal is double the background signal. Silk Fibroin Intradermal Safety In addition to anti-influenza indirect ELISAs, anti-silk ELISAs were performed to assess the immune response to the biomaterial itself. Silk fibroin is FDA-approved for use as a suture material and has been demonstrated to be biodegradable into non-inflammatory amino acids and peptides27-30. However, in the microneedle delivery format the silk material and vaccine are co-presented to dermal dendritic cells. For the devices to remain efficacious in the clinical setting, the coating formulation of silk with vaccine must not generate any adverse reaction upon primary or secondary antigen presentation. The indirect ELISA protocol for anti-influenza response was modified for anti-silk IgG response by coating microtiter plates with 4 µg/mL silk fibroin (30 min extraction). Serum samples were pooled from the 5 animals and then used to determine if any immunological memory against the biomaterial is raised in the murine models when an influenza response is also raised. Pooled sera was also run on the anti-influenza ELISA for direct comparison of IgG titers to provide a relative measure of safety/immunogenicity. Immunogenicity of Chimeric C. difficile Vaccine and Protective Efficacy The immunogenicity of the cTxAB vaccine for C. difficile was first assessed through ELISA. Blood samples were collected from all mice 1-day prior to each of the scheduled immunizations as well as 3weeks after the final immunization (prior to spore challenge). Indirect ELISA was performed by coating plates with C. difficile toxin A or toxin B proteins followed by a blocking step45. Anti-toxin A and antitoxin B murine polyclonal antibodies were used as a positive standard. Dilution series of the standards and serum samples were prepared and added to the microtiter plates. Following incubation for 2 h, secondary antibody was added and incubated prior to development with TMB. The assay was stopped after 10 min of development and absorbance was read at 450 nm. Endpoint IgG titers were calculated for each animal and compared after each immunization to assess the antibody response to the chimeric vaccine delivered via three routes of administrations. In addition to the serology workup, measurements of the protective efficacy of the chimeric C. difficile vaccine were performed. C. difficile UK6 isolated in the United Kingdom was used in this study, and sporulation of the C. difficile UK6 was induced as previously described50-53. All the mice were treated with the antibiotic cocktail (kanamycin, gentamicin, colistin, metronidazole, and vancomycin) and clindamycin as previously described before spore inoculation54. The mice were then challenged orally with 1 x 106 spores of C. difficile UK6, and were monitored for one week post-challenge. Animals were examined daily for diarrhea and weight loss, indicative of infection by the pathogen and incomplete protection with the vaccine. Immunogenicity of Shigella Fusion IpaDB Vaccine Female BALB/c mice (4-6 weeks, Charles River Labs, Wilmington, MA) were immunized three times with fusion IpaB/IpaD antigen at two-week intervals to examine the serum and mucosal antibody responses generated via three administration techniques (transdermal microneedle, ID injection, intramuscular (IM) injection). To assess the immunogenicity via each of these techniques, serum was collected prior to administration and 13 days after each immunization. These serum samples were examined via indirect ELISA to determine IgG endpoint titers and measure any attenuation in response due to route of administration. Similarly, mucosal IgA responses were evaluated via ELISA after collecting fecal samples, saliva swabs, and vaginal washes from animals at the final time point.


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RESULTS Fabrication of Vaccine-Coated Silk Microneedles We previously reported a simple, non-cleanroom-based process to fabricate silk fibroin microneedles and demonstrated successful delivery of model therapeutics into human cadaver skin with these devices22. Here, we sought to demonstrate the compatibility of such coated silk fibroin microneedle devices with both discovery-stage and commercial vaccine products while assessing their performance and efficacy in vivo. To prepare the vaccine-coated silk microneedles, PDMS molds used in our previous work were filled with silk fibroin solution and centrifuged for 20 min at 4°C and 5,000 rpm to force solution into the needle cavities of the mold22 (Figure 1, A). Additional silk fibroin solution was added to the molds to ensure the formation of a thick film backing, and molds were dried for 2-3 days under ambient temperature and humidity. Silk fibroin microneedles were carefully removed, water annealed for 24 h, and manually trimmed to remove the edges of the silk film backing24. The devices were dried for an additional 24 h prior to the vaccine coating process. To ensure consistency of the vaccine coatings with each of the vaccine antigens used in these studies, silk microneedle patches were coated in a two-step process. First, the microneedle surfaces on the devices were pre-wet with deionized water for 5-10 min (Figure 1, B). Vaccine coating formulations were prepared through dilution of the antigen solutions to the desired final concentration using silk stock solution, adjuvant stock solution, and deionized water. An appropriate volume of these vaccine-containing formulations was coated onto the wet microneedle patches to achieve the desired antigen dose and these devices were dried for 24 h under ambient conditions, protected from light. Vaccine-coated silk fibroin microneedles were then used within 24 h for animal immunization studies to minimize any loss during storage. Influenza Vaccine Transdermal Immunogenicity To assess the efficacy of silk fibroin microneedles for transdermal vaccination with a commercial influenza vaccine (Fluzone Quadrivalent 2014-2015, Sanofi Pasteur, Swiftwater, PA), we prepared microneedles with up to a 12 µg dose of vaccine formulated in a 1% (w/v) silk fibroin solution coating. These microneedles were applied to pre-shaved and cleaned dorsal skin of BALB/c mice and were fixed with adhesive dressing (Tegaderm, 3M, St. Paul, Minnesota). After 24 h of application, microneedles were removed and we observed no signs of local dermal sensitization (more information in SI). Additional mice were injected subcutaneously in the same dorsal region as a control. This SC route was chosen over ID as the vaccine concentration could not be adjusted and we expected the volume needed for 12 µg would be better tolerated by mice SC than ID. Mice were re-immunized two weeks after the initial immunization. Serum was collected prior to each immunization as well as two weeks post-boost. We examined the formation of IgG antibodies against the commercial influenza vaccine via ELISA, observing a delayed IgG response from the microneedle groups relative to the subcutaneously injected group (Figure 2). Prior to boost, none of the microneedle groups displayed significant increases in IgG titer. After mice received a second microneedle administration, a subset of animals began to produce antiinfluenza IgG antibodies; however, this response was inconsistent in the 6 µg and 12 µg dose groups. Roughly half of the animals in these groups displayed a 100-fold increase, greater than the response to subcutaneous injection (difference in means between these groups was not statistically significant, one-way ANOVA, F[2,12] = 0.4931, p = 0.6226). All statistical analysis for influenza, Shigella, and C. difficile was performed using GraphPad Prism 7. This inconsistency in response was not observed in the subcutaneous injection group, and while it may indicate some difficulty with reproducible administration in an experimental setting, these results confirm the ability to deliver commercial influenza vaccine transdermally using silk fibroin microneedles. Silk Fibroin Intradermal Safety Microneedles were coated with influenza vaccine and 1% silk fibroin to impart temperature stability to the antigens during the air-drying process and storage. However, we performed no additional post-



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processing on the patches and expected the silk to remain soluble, allowing it to be co-delivered with influenza vaccine into the skin of mice. While silk fibroin is an FDA-approved biomaterial and has been previously characterized for immunogenicity, we examined the extent of immune response to the material relative to influenza antigen when delivered intradermally27-30, with the caveat that the silk used here was generated under laboratory conditions and not under more rigorous GLP or GMP conditions. The same indirect ELISA used to assess the level of anti-influenza IgG antibodies in mouse serum was modified by changing the coating antigen to silk fibroin solution of the same molecular weight distribution (60 min extraction). Serum from the final collection was pooled for the 5 animals per positive-responding immunization group (6 µg microneedle-immunized, 12 µg microneedle-immunized, 12 µg subcutaneously injected) and run on the parallel ELISAs for anti-silk and anti-influenza titer determination due to sample volume constraints. We found that the influenza antigen is immunodominant in the coating formulation, generating significantly higher titers at each delivered dose than silk fibroin (Figure 3). While the IgG response to silk remained close to pre-bleed titer values, the increase in titer as the ratio of silk to antigen increased was found to be statistically significant. This result is not unexpected as the total dose of silk delivered in this formulation (1,000 µg) is orders of magnitude higher than influenza antigen. In addition, the increase in anti-silk titer (~2X) is much lower than that reported for gelatin in commercial measles, mumps, and rubella vaccine (>30X)55. The anti-silk response could be removed through reduction of the soluble silk protein in the formulation or post-processing to render the protein insoluble. Overall these results suggest that silk fibroin is generally safe for ID delivery and minimally immunogenic. Immunogenicity of Chimeric C. difficile Vaccine and Protective Efficacy To examine the efficacy of a novel C. difficile vaccine when delivered transdermally, we prepared silk fibroin microneedles coated with 10 µg of cTxAB vaccine and 1 µg of the adjuvant dmLT. These devices were administered to C57BL/6 mice three times at two-week intervals and compared with dose-matched ID and IP injections (IP contained alum adjuvant instead of dmLT). Serum was collected 13 days after each immunization to examine the formation of antibodies against toxin A and toxin B. We observed that the microneedle delivery group successfully delivered the vaccine and produced a robust response against toxin A (Figure 4, top). Although the toxin B response from the microneedle delivery group was significant relative to naïve mice, it was slightly attenuated relative to the ID and IP delivery routes (Figure 4, bottom). The administration of C. difficile vaccine using silk microneedle patches also led to a delayed response against toxin A, with significantly lower titers generated after the first immunization than ID and IP injections despite similar titers after the second and third administration. In addition to evaluating the antibody titers against toxins A and B, we challenged the immunized and naïve mice with toxigenic C. difficile to assess protection following microneedle-based vaccination. Three weeks after the final immunization, we challenged the mice with 1x106 spores of C. difficile strain UK6 and monitored their health for one week post-challenge. Specifically, we examined cages daily for diarrhea and weighed mice to determine weight loss, general signs of successful infection by the spores and incomplete protection of the vaccine. We observed that all of the naïve mice lost weight two days after the challenge, while the animals that received vaccine via ID or IP injection did not lose weight (Figure 5, top). The microneedle-vaccinated group lost half as much weight as the unvaccinated group but recovered more quickly. This recovery is observed in the persistence of diarrhea in all naïve mice for three days, with only 2 mice in the microneedle group displaying symptoms one day post-challenge and 1 mouse after two days (Figure 5, bottom). While the vaccine was not as efficacious as ID or IP injection when delivered transdermally with microneedles, it did provide meaningful protection against C. difficile challenge in a majority of the animals. Immunogenicity of Shigella Fusion IpaDB Vaccine The final vaccine we evaluated on the silk fibroin microneedle devices was a fusion protein of Shigella protective antigens IpaD and IpaB. As these antigens are conserved across strains of Shigella bacteria, a vaccine utilizing these antigens could provide broad protection against shigellosis. We fabricated silk


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microneedles and coated them with the standard formulation of 1% silk fibroin containing 30 µg of the fusion protein and 1 µg of the adjuvant dmLT. These microneedles were administered to the shaved and cleaned dorsal skin of BALB/c mice and left applied for 24 h. This delivery method was compared to ID and IM injection using 10 µg of fusion protein and 1 µg of adjuvant. After performing a three-dose immunization series, we collected blood, saliva, feces, and vaginal rinsate to assess local and systemic protection via antibody production. Specifically, ELISA was used to determine IgG titers in serum against IpaB and IpaD antigens and IgA titers in feces, saliva, and vaginal wash. Injection of the IpaDB vaccine generated strong IgG responses against both IpaB and IpaD antigens after one dose while microneedlevaccinated groups displayed significant antibody titers after two doses (Figure 6, A and B). However, the IgG response from the microneedle group against IpaB and IpaD was lower than the response produced through vaccine injection after three doses. The mucosal IgA against IpaB and IpaD was detected after IpaDB immunization (Figure 6, C and D), however there was no significant difference among the immunized groups. These results imply that silk microneedles successfully delivered the fusion protein vaccine but did not generate an equivalent response to the injected groups.



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DISCUSSION The use of microneedles for the transdermal delivery of macromolecules has gained significant momentum over the past decade, with a number of natural and synthetic materials being investigated in these delivery systems7, 12, 56-58. For successful clinical acceptance of these devices, microneedles and their constituent materials must meet specific product criteria including: processing techniques amenable to formulation with sensitive macromolecules, minimal premature degradation of material or macromolecule during storage, adequate mechanical strength to penetrate the stratum corneum, and dermal biocompatibility59. Silk fibroin is a unique biomaterial that can be added to the growing list of materials investigated for these product criteria as a transdermal delivery device. The fibroin protein offers appealing characteristics for a microneedle device as it can be aqueously-processed with controllable degradation, is mechanically robust and biocompatible, and can impart thermostability to therapeutic payloads23-24, 26-34. Here, we have built upon our previous work to fabricate silk fibroin microneedles by coating them with vaccine formulations against three pathogens and evaluating their efficacy. The targeting of the immune cell population of the skin through microneedle vaccination has been well described with significant benefits reported in terms of enhanced antibody and cellular response3-6, 9. In the present work, we observe successful silk microneedle delivery of influenza, Clostridium difficile, and Shigella vaccine antigens through the generation of humoral immune responses. While promising, the specific IgG, IgA, and protective responses were generally lower than the injected controls with the same dose formulation. We attribute this to the incomplete elution of the full dose of antigen off the patch. During the vaccine coating process, some antigen is deposited onto the depressions between adjacent microneedles. This area is not exposed to the interstitial fluid in the epidermis during administration, leading to incomplete doses delivered. Most coated or dissolving microneedle systems are already limited to maximum doses approaching 10s of micrograms of therapeutic, so the delivery efficiency from silk microneedles is important39, 60-61. As we did not quantify residual antigen on the silk microneedle devices, we estimate from surface area calculations that a maximum of 47.7% of coated antigen was available for delivery. This points to the importance of quantifying antigen on the silk microneedle patches at each stage of processing and after deployment. Our serology results can be correlated to the expected coating dose but would benefit from information regarding coating efficiency. While the coating process is reproducible to the extent that it is volume-metered, we cannot evaluate potential antigen loss during airdrying or storage which may contribute to the lower immune responses observed from microneedle groups. Furthermore, as the amount of antigen coated on the patches changes, the thickness of the coating is also expected to change and may impact the insertion force required to penetrate the stratum corneum. Standardizing the thickness of the microneedle coating with an inert solid, such as a sugar, would allow us to mitigate this concern when analyzing serology results. To advance this silk microneedle platform to further preclinical studies, this coating and delivery characterization will be critical. The immune responses from coated silk microneedles provide evidence for dose sparing as the actual dose delivered was lower than the coated dose. Similar delivery systems have reported 3-5X dose sparing for similar influenza antigens43, 62. Through these initial efficacy studies, we have demonstrated that silk microneedles show promise for the delivery of vaccine antigens. The devices were worn by mice for 24 h to ensure complete delivery of antigen available to interstitial fluid and to maximize exposure to silk for safety assessment. With no adverse reactions observed following this prolonged exposure time, we are encouraged by the performance of silk microneedles. Other groups have examined transepidermal water loss following microneedle-mediated delivery to quantify the porosity/healing after administration63-64. This method will help supplement our safety data with silk microneedles while also enabling improvement to the microneedle application to the backs of mice. Microneedles were pressed into the skin of the animal and held there by the researcher’s fingers for a few minutes, resulting in some variability in the overall area of contact with the skin and another source of incomplete dose delivery. The further optimization of the physical application will ensure more reproducible and complete antigen


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delivery. Like other materials, silk fibroin has advantages and disadvantages, but with improvements to decrease wear time, improve characterization of coating and delivery efficiency, and reduce fabrication time we expect the material will remain a viable option for microneedle delivery devices.



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CONCLUSIONS With the need to develop low-cost and efficacious strategies to improve vaccination coverage, microneedles have emerged as a leading solution due to their simplicity and ability to be selfadministered. As a result, many materials have been evaluated with varying degrees of success in this delivery format. Silk fibroin protein possesses characteristics such as mechanical strength, all-aqueous processing, and biocompatibility that make it a suitable material choice for microneedle devices. In the present study, we have demonstrated initial efficacy with influenza, Clostridium difficile, and Shigella vaccines delivered transdermally using silk microneedles. We also found silk to be minimally immunogenic in comparison to the antigen payloads and conclude that the material is safe for ID administration. While there are improvements to be made to accelerate fabrication and better characterize the coating and delivery efficiency, the successful generation of humoral immune responses against all three vaccines is encouraging. The silk fibroin material combines some of the processing and biocompatibility advantages of a dissolving microneedle system with the product stability and mechanical strength of coated insoluble microneedle devices. Furthermore, the protein can be formulated to thermostabilize biomacromolecules which could enable refrigeration-free vaccination devices. This would represent a significant step towards increasing global vaccine coverage, and could be applied to other therapies as well. With new cancer vaccines being developed to focus on T-cell responses, microneedleenabled delivery to the dermis may result in improved responses over traditional routes of injection. From the present work, we conclude that silk fibroin microneedles represent a promising strategy for transdermal immunization, but will require further optimization and characterization to reach their potential.


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FIGURES

Figure 1. Fabrication process for vaccine-coated silk fibroin microneedles. (A) PDMS molds used in previous studies22 were filled with a volume of silk solution and centrifuged to force material into the needle voids. Additional silk was added to produce a robust backing after 2-3 days of drying under ambient conditions. Silk microneedles were de-molded, water annealed, and trimmed to produce the final uncoated constructs. (B) To evenly coat the surface of silk microneedles with vaccine-containing solution, the devices were pre-wet with droplets of water. After 2-5 min of dwell time, the water fully wet the microneedle surface and was pipetted off before the addition of antigen solution. The coatings were dried overnight prior to administration in vivo.



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Figure 2. Influenza vaccine delivery using silk fibroin microneedles generates measurable IgG titers post-boost at medium (6 µg) and high (12 µg) doses. Serum was collected from animals preimmunization, post-primary immunization and post-boost and was analyzed via ELISA for IgG titers against the influenza antigens. Although attenuated mean responses were observed for these microneedleimmunized (MN) groups relative to the subcutaneously injected (SC) group, higher anti-influenza IgG titers were generated in a subset of these animals relative to the SC group. Displayed are endpoint titers for each animal, with geometric mean and 95% confidence interval calculated for each group (n = 5). The differences in mean endpoint IgG titers for 6 µg MN, 12 µg MN, and 12 µg SC groups were not statistically significant (one-way ANOVA, F[2,12] = 0.4931, p = 0.6226).


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*

*

Figure 3. Influenza antigen is immunodominant in silk microneedle coating formulation. As a foreign protein, silk is degraded into non-inflammatory products and is known to be biocompatible. Antisilk IgG-specific responses were examined post-boost on pooled sera from 5 animals in groups demonstrating an IgG response to influenza vaccine. For the microneedle patches co-delivering influenza and silk, statistically higher anti-influenza IgG titers than anti-silk were observed, indicating the immunodominant nature of the influenza epitopes (Student’s t-test; 6µg: p = 0.003, df = 6; 12µg: p < 0.001, df = 6). As the ratio of silk protein to influenza antigen increased, a statistically significant increase in titer was observed although values remained close to pre-bleed levels (one-way ANOVA, F[2,6] = 269.1, p < 0.001). MN: microneedle-immunized; SC: subcutaneously injected



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Figure 4. Antibody response to chimeric toxin C. difficile vaccine candidate. Mice (n =5 per group) were immunized 3 times at two week intervals with: no vaccine on a silk microneedle patch (CP), vaccine patch with 10 µg chimeric toxins and 1 µg dmLT adjuvant (VP), intraperitoneal injection with 10 µg chimeric toxins and alum adjuvant (IP), and intradermal injection with 10 µg chimeric toxins and 1 µg dmLT adjuvant (ID). Serum samples were collected prior to each immunization and analyzed via ELISA for the presence of IgG against C. difficile toxin A and toxin B. In comparison to injection controls, silk microneedles produced a comparable response for toxin A and a slightly weaker response against toxin B. A. serum anti-Tcd A IgG titer; B. serum anti-TcdB IgG titer. Tukey's multiple comparisons test. *p

Silk Fibroin Microneedles for Transdermal Vaccine ... - ACS Publications

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