Ph.d.-forsvar af Stephanie Ingemann Bisgaard

Principal supervisor: Professor Stephan Sylvest Keller, DTU Nanolab
Co-supervisor: Senior researcher Katrine Lindholm Bøgh, DTU Food

Examiners:
Associate Professor Line Hagner Nielsen, DTU Sund
Professor Urs Otto Häfeli, Pharmaceutical Sciences, UBC
Professor Per Stahl Skov, SDU & Head of Reflab Aps

Chairperson at defence: Associate Professor Paul Kempen

Abstract

The prevalence of allergies is increasing worldwide and is a global burden for the healthcare sector. The skin prick test (SPT) is a common method for screening for allergies. It takes 15-20 min and requires a manual readout by a healthcare professional. However, the response is only qualitative, not quantitative. 
The aim of the InstaPatch project, which this PhD thesis is a part of, was to improve on the SPT and develop a novel test for fast and quantitative allergy diagnostics in the skin. The InstaPatch is envisioned as an intradermal electrochemical biosensor based on microneedles (MNs) which are sub-mm structures that act like small needles piercing the skin. The first aim of the PhD project was to ensure the mechanical stability and penetration ability of in-plane solid silicon MNs. The MNs should cause as little tissue damage as possible while ensuring sufficient penetration depth to reach the upper dermis skin layer. Five MN shapes with varying dimensions were tested in skin-simulating hydrogels, excised Brown Norway (BN) rat (rattus norvegicus) skin, and porcine ear skin samples using a Texture Analyser to obtain force displacement graphs for the penetration. The MN shape and dimensions were optimized to minimize penetration force while maintaining mechanical stability. Induced tissue damaged was examined using histology and the triangular shape was found to cause the least amount of tissue damage. The optimized MNs were a hybrid between a triangular and pencil shape exhibiting good mechanical stability and penetration ability while providing enough surface area for coating and future electrochemical measurements. The optimized MNs were tested in porcine skin and human breast skin samples to evaluate their mechanical stability and penetration ability. The MNs were 1000 μm long, 400 μm wide, and 180 μm thick. The second aim of the PhD project was to deliver an allergen by MN insertion to initiate a local allergic reaction. First, MNs were coated using dip-coating with fluorescent-labeled molecules for visualization and quantification of delivery. It was shown that MNs could be coated with fluorescent-labeled molecules and delivery was confirmed by cryostat histology and fluorescence microscopy. 
However, by measuring the remaining, fluorescent-labeled coating found on the MNs after insertion, the delivered amounts were found to be exceedingly small and hard to quantify. The final aim of the PhD project was to deliver an allergen in sensitized BN rats using allergen-coated MNs and confirm a local allergic reaction. In vivo experiments were performed. One used peanut protein extract (PPE)-sensitized BN rats and another used birch pollen extract (BPE)-sensitized BN rats. MNs were coated with the peanut allergen Ara h 2 at various concentrations for delivery in PPE-sensitized rats while other MNs were coated with BPE or the birch pollen allergen Bet v 1 in various concentrations for delivery in BPE-sensitized rats. 
Coatings with compound 48/80 were included as positive control and phosphate buffered saline as negative control. Intradermal (i.d.) injections of the same allergen solution concentrations were included as a second control for the coated MNs. The allergic response within the skin was evaluated by intravenously injecting Evans blue into the tail vein to visualize blood accumulations. It was not possible to measure a significant reaction in sensitized BN rats when the delivery was facilitated by the MNs. 
However, some promising, but sub-optimal dose-response curves were measured for rats receiving i.d. injections. As a last step, delivered Ara h 2 was attempted recovered in homogenized biopsy samples from the BN rats. Ara h 2 could be measured in the homogenized samples for delivery from the highest i.d. injection concentration, however, not from the MNs.