Iontophoresis as a non-invasive enhancement technique for the administration of drugs across biological membranes

Iontophoresis, a technique that consists in applying low density current to a membrane, has been widely investigated in order to enhance the permeation of drugs through different biological barriers such as the skin, the buccal mucosa and the sclera in order to obtain a systemic or local (in case of trans-scleral administration) effect without the need of an injection. The aim of this thesis was to investigate the effect of iontophoresis on these three barriers, considering the different state of the art for each of them. Concerning trans-scleral iontophoresis, there are many studies with small molecules, and some on neutral and negatively charged macromolecules, while there’s a lack of data on positively charged macromolecules, which can be useful for the treatment of different posterior segment eye diseases. Cytochrome c (12.4 kDa, charge +7.9 at pH 8.2) was chosen as a model molecule. Passive transport of cytochrome c was evaluated across the sclera and the bilayer choroid-Bruch’s membrane. The affinity of cytochrome c for melanin was measured by means of in vitro binding experiments. Iontophoretic permeation was studied as a function of donor concentration (5 to 70 mg/ml) and current density (1.51 to 5.83 mA/cm²), and the contribution of electroosmosis on cytochrome c transport was determined using a high molecular weight fluorescent dextran (FD-150) as neutral marker. Moreover, also the possibility of a fine tuning of the release was assessed. The results showed that cytochrome c permeated across isolated sclera with a permeability coefficient of about 2.5*10-6 cm/s, comparable to molecules with a similar radius. Choroid-Bruch’s membrane bilayer was an important barrier to permeation, also because of the presence of melanin in this tissue. Iontophoresis (2.92 mA/cm²) enhanced cytochrome c permeation across the sclera at all the concentrations tested, increasing about 10 times the amount permeated after 2 hours. The effect was proportional to current density. Iontophoretic experiments performed with FD-150, an electroosmotic flow marker, demonstrated that the presence of cytochrome c dramatically reduced the EO flow and that, despite the high molecular weight, the main mechanism for cytochrome c iontophoretic permeation is electromigration. Finally, experiments performed with an alternation of current-assisted and passive permeation showed that a modulation of the release was possible. Buccal mucosa is an interesting alternative site to administer drugs, since it can avoid first pass effect. However, permeation is limited by the barrier properties of the buccal epithelium. Thus, iontophoresis may be useful to promote the permeation of drugs. Recently, pig esophageal epithelium has been proposed as a model for buccal epithelium, since it is easier to obtain and separate, and it has shown to be similar in structure and lipid composition compared to buccal tissue. There are limited data about buccal iontophoresis, so the aim of this part of the project was to characterize the properties of esophageal epithelium under the application of electrical current. Sumatriptan succinate was chosen as a model, because of its well characterized behavior during transdermal iontophoresis. The effect of raise in current density (0-5.83 mA/cm2) and of concentration in the donor solution were assessed. The increase in the amount permeated was proportional both to the increase in current and in donor concentration. On the other side, data in literature on transdermal iontophoresis are abundant, on both small molecules and macromolecules. In this case, we studied the effect of iontophoresis compared to laser microporation, another physical enhancement technique, on the permeation and deposition of Thymosin α1. This is a 28 amino acids peptide used to treat different pathological conditions and as an adjuvant for vaccination. Even though the amounts permeated were probably not enough to elicit a systemic response from the immune system, iontophoresis allowed higher permeation in a shorter period of time compared to laser microporation.