Doctoral thesis

Cytochrome c bioconjugation – Creating an iontophoretic shuttle for the non-invasive delivery of proteins

Number of pages208
Imprimatur date2023
Defense date2023

Topical drug delivery is one of the most suitable routes of administration to manage skin conditions. It is defined as the application of a dosage form on the surface of the skin to deliver the drug to its site of action at therapeutic levels to ensure a response. Transdermal drug delivery is usually defined as the process of solute transport through the various layers of the skin into the systemic circulation. Once distributed, the drug can then produce the desired therapeutic effect. This route of administration has several advantages: it reduces the exposure of substances to extreme environments such as the gastrointestinal tract and avoids the effect of first-pass metabolism. However, the barrier function of the skin is mainly ensured by the stratum corneum, the outermost layer of the skin. Nevertheless, strategies to overcome the skin barrier function are constantly being investigated to allow the delivery of higher molecular weight and more hydrophilic drugs, such as peptides and proteins. This thesis will mainly evaluate cutaneous and transdermal delivery using iontophoresis, a non-invasive needle-free energy- powered technique. Iontophoresis is a physical enhancement technique involving the application of a low electrical potential to enhance the non-invasive delivery of ionisable species into and through biological membranes such as the skin.

Iontophoresis has been well described for the transdermal delivery of low molecular weight drugs. In subsequent studies, higher molecular weight compounds, such as peptides and proteins, were also studied. However, although these studies demonstrated the feasibility of using transdermal iontophoresis to deliver proteins non-invasively through intact skin, they also showed that simple descriptors such as molecular weight, charge and electrical mobility alone did not accurately predict transdermal iontophoretic transport.

The objectives of this thesis were to describe an experimental study on the development of an iontophoretic drug delivery platform using cytochrome c (CytC). CHAPTER A will first describe the iontophoretic delivery of SMT3 (a small Ubiquitin-like protein), a negatively charged model protein. This is a report on the use of anodal iontophoresis to deliver an anionic protein, not widely described in the literature. It was possible to reveal that electroosmosis was the main transport mechanism responsible for its anodal iontophoretic delivery. The results also suggest a high degree of degradation of the protein once in contact with the skin. Then, a head-to-head comparison of the transdermal delivery of the latter protein was evaluated by conjugation as a fusion protein to the CytC shuttle. The present study is the first to demonstrate the feasibility of using iontophoresis to deliver a protein as a bioconjugate to the skin as well as through intact skin. The results suggest a 10.6-fold increase in SMT3 deposition when conjugated to CytC with no significant difference in transdermal delivery. This was possible although the extreme impact of the physicochemical properties of the ~14 kDa anionic protein on the cationic shuttle of the cytochrome, despite the fact that these characteristics are essential for its electrotransport.

To complement these results and further develop a CytC shuttle delivery platform, CHAPTER B was focused on platform design and evaluation of fluorescein bioconjugation.

Citation (ISO format)
FARO BARROS, Jonathan. Cytochrome c bioconjugation – Creating an iontophoretic shuttle for the non-invasive delivery of proteins. 2023. doi: 10.13097/archive-ouverte/unige:176213
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