Reducing Allergic Airway Inflammation with High-Density Microprojection Array Skin Patches
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Reducing allergic airway inflammation with high-density microprojection array skin patches Nicole Monica Dawn van der Burg Bachelor of Science (Hons I) in Biochemistry and Genetics A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Australian Institute for Bioengineering and Nanotechnology I. Abstract Inflammation of the airways can be life threatening for those with T helper type 2 (Th2)-mediated allergies or allergic asthma. Despite the increasing prevalence of allergic diseases, there are no current vaccinations to prevent allergies. Desensitisation via allergy immunotherapy (AIT) is the only disease-modifying treatment available. This involves slowly building up a specific-tolerance to the allergic substance (allergen) through either subcutaneous injection or oral absorption. Yet allergy patients rarely undertake AIT as is it costly, time consuming and can increase the risk of life threatening anaphylaxis. These discouraging factors arise from the poorly optimised targeting of the current routes of AIT that do not target immunotherapy primarily to tolerant-inclined cells in order to maximise an allergen-specific tolerance response. Building allergen-specific tolerance is often similar either before (vaccination therapy) or after (desensitisation therapy) allergen sensitisation. Specific-tolerance is established when tolerant- inclined cells, such as skin epidermal Langerhans cells (LCs), present allergens to the immune system alongside tolerant signalling. Targeting the therapy to the skin has been notoriously challenging with current needle and syringe or topical patch methods. However, microprojection array (MPA) patches have begun to demonstrate rapid delivery of vaccine therapeutics as well as specific cell targeting into the upper layers of the skin. Immunisation with high-density (i.e. above 1,000 (1k) projections (p)/cm2) dermal-targeted MPAs (dMPA) have previously been shown to induce pro-inflammatory and specific-IgG responses at a fraction of the injectable dose. As an increase in IgG correlates with a decrease in IgE during AIT, I hypothesised that the dMPA-therapy could specifically inhibit IgE in a desensitisation model. After initial results, I further hypothesised that a MPA that delivered allergen primarily to the epidermal LCs (eMPA) would prevent Th2 inflammation in a vaccination model, similar to that seen in a healthy immunity against allergens. Therefore, different designs of dMPAs were tested in desensitisation therapy and eMPAs in preventative vaccination therapy with an ovalbumin (OVA)- based, IgE-mediated, airway hypersensitivity mouse model. First, conical-shaped dMPAs of two densities (21k or 10k p/cm2) delivered OVA at application energies of either 170 mJ or 100 mJ to the skin of OVA sensitised BALB/c mice. Desensitisation with the very high density 21k-dMPA significantly increased unwanted Th2 responses including airway eosinophilia and anti-OVA IgE. Based on these results, the dMPA was tailored to reduce the impact on the skin by halving the projection density to 10k and using an application energy of 100 1 mJ. Despite the increased anti-OVA IgG1 and IgG2a, the anti-OVA IgE continued to persist. Yet, both airway eosinophilia and the level airway mucus was significantly reduced at day 87 in 10k- dMPA-100 mJ treated mice. To date, the association between repeated OVA-dMPA treatments (without adjuvants) on preventing airway eosinophilia and mucus has not been reported. Second, after establishing a strong correlation between decreased dMPA density and Th2-mediation of the immune response, I aimed to test a low inflammatory eMPA in vaccination. To date, no MPA design was reported to successfully target therapeutics to the epidermis of mouse skin. To ensure a shallow (~15 µm) but consistent epidermal delivery, projection tips were widened from the pointed conical shape to a slit-like shape, increasing the tip surface area. Slit-shaped eMPAs applied at 30 mJ resulted in significantly less erythema and epidermal inflammation than dMPAs. Analyses of the inguinal skin draining lymph nodes found that eMPAs (with or without OVA) significantly increased LC migration, but not dermal dendritic cell migration. This is the first report of MPAs targeting the mouse epidermis and preferentially activating LC migration. Third, various doses of OVA delivered by low-impact eMPAs were applied to naïve mice to test prevention of Th2 airway inflammation. Compared to the positive control (80-200 µg), the eMPA prevented airway eosinophilia in up to 60% of mice and anti-OVA IgE in 75% of those mice with a significantly lower dose (0.4 µg). Additionally, mice vaccinated with eMPAs had significantly less airway mucus and obstructions even when challenged with a chronic sensitisation model. Therefore, unlike other transdermal quick-delivery devices, the eMPA showed promise in preventing airway hypersensitivity by delivering just the allergen. In conclusion, by reducing the impact on the skin, both dMPAs and eMPAs applied to mouse skin reduced airway inflammation. Only desensitisation with 10k-dMPAs at 100 mJ reduced airway eosinophilia, suggesting high densities and/or application energies are too Th2-mediated to prevent eosinophilia. Similarly, only vaccination with fewer eMPA repeats applied with lower doses prevented airway inflammation, suggesting that eMPA vaccination is dependent on the level of impact on the skin and dose delivered. These findings show promise for the future use of high- density MPAs as a skin deliver device of allergen against airway hypersensitivity but highlights the importance of understanding the new variables such as application energies and overall impact the MPA design has on the skin during MPA delivery of allergen. 2 II. Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. 3 III. Publications during candidature A. Publications included in this thesis No publications included. B. Submitted manuscripts included in this thesis Literature review: No manuscripts submitted for publication Chapter 4: No manuscripts submitted for publication Chapter 5: No manuscripts submitted for publication Chapter 6: Manuscript submitted included as Part A. 1. Nicole M.D. van der Burg, Alexandra C.I. Depelsenaire, Michael L. Crichton, Paula Kuo, Simon Phipps and Mark A. Kendall. Langerhans cell-targeted microprojection patch to deliver ovalbumin to the epidermis of mouse skin. Submitted to the Journal of Controlled Release on the 3rd of December 2018. Chapter 7: No manuscripts submitted for publication Chapter 8: No manuscripts submitted for publication C. Conference abstracts (oral presentations) 1. N.M.D. van der Burg, et al.: Preventing allergic airway eosinophilia with a Langerhans cell targeted microprojection patch. Presented at European Academy for Allergy and Clinical Immunology, Germany (2018). Appendix 10.1 2. N.M.D. van der Burg, et al.: Targeting allergy immunotherapies to the epidermis, for improved allergy prevention. Presented at BioNano Innovation, Australia (2017). Appendix 10.2 4 3. N.M.D. van der Burg, et al.: Targeting therapies to the epidermis of the skin. Presented at ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, Australia (2017). Appendix 10.3 4. N.M.D. van der Burg, et al.: Improved allergy desensitisation by targeted delivery to the immune cells within the skin strata. Presented at the Australian Institute for Bioengineering and Nanotechnology, Australia (2016). Appendix 10.4 5. N.M.D. van der Burg, et al.: Setting up a working mouse model to test alternative skin targeted allergy desensitisation immunotherapy methods. Presented at Translational Research Institute Immunology Series, Australia (2015). Appendix10.5 D. Conference abstracts (poster presentations) 1. N.M.D. van der Burg, et al.: Preventing allergies with an epidermal microprojection patch. Presented at Australasian Society for Immunology