Controlled Ultrasound Erosion for Transdermal Delivery and Hepatitis B Immunization

Abstract

Although ultrasound is effective for transdermal delivery, it remains difficult to control the position, shape and size of localized skin transport regions. We developed an ultrasound erosion protocol to generate a single-site, circular delivery region with controlled size at the center of patched skin. We found that (i) shorter ultrasound pulses (25 cycles) with higher pulse repetition frequency (4 kHz) and higher peak negative pressure (17.0 MPa) resulted in larger (0.995 mm²) and deeper (∼300 µm) skin delivery regions with a higher success rate (94.44%); and (ii) temperature elevation of the skin increased with ultrasound exposure time, with a 30-s safety threshold. Furthermore, we found that hair follicles decreased the delivery controllability of ultrasound erosion. Therefore, we selected the skin of the hind legs of mice without dense hair follicles to deliver more than 1 μL of vaccine solution and successfully elicit immune responses against hepatitis B surface antigen.

Introduction

Vaccination is the most effective preventative measure against infection and infection-related mortality (Childs et al. 2018). Conventional vaccinations via needle injection must be administered by well-trained medical professionals. Transdermal immunization is an attractive approach based on the concept of self-administered vaccination. Apart from its ease of use, it has been suggested that transdermal immunization could enhance immune responses because the skin is an advanced immune system with large populations of immune cells in its functional layers: for example, Langerhans cells and dendritic epidermal T cells reside in the epidermis, and macrophages, T cells and mast cells reside in the dermis (Tong et al. 2015). Therefore, antigens delivered by transdermal immunization could be presented more effectively to the body's immune system. Because the passive diffusion of molecules larger than 500 Da is limited by the stratum corneum of the skin, various methods have been developed to achieve transdermal delivery, including iontophoresis, electroporation and ultrasound- and laser-mediated transdermal delivery (Lee et al. 2018).

Ultrasound-mediated transdermal delivery, first reported in 1989, has proven to be effective for the delivery of a large number of drugs (Levy et al. 1989). Although the mechanisms underlying ultrasound-mediated transdermal delivery are not fully understood, it is generally accepted that two physical effects (i.e., thermal and cavitation effects) contribute to the permeability enhancement of ultrasound-treated skin. It has been suggested that the thermal effect, which results from the attenuation and absorption of ultrasound energy by skin, plays a minor role (∼25% of the total enhancement) in transdermal delivery (Merino et al. 2003), whereas the cavitation effect, which results from the violent oscillation and collapse of gas nuclei located near or in the skin, is the main contributor to enhanced skin permeability (Polat et al. 2011). Therefore, low-frequency ultrasound (20–100 kHz), which is the most effective method in fostering cavitation events, has been widely used in transdermal drug delivery (Ueda et al. 2009). In addition, cavitation agents (e.g., microbubbles and porous resins) are added to the coupling medium between the skin and the ultrasound transducer to increase the total number of ultrasound-triggered cavitation events (Park et al., 2012, Terahara et al., 2002). Despite these efforts to enhance cavitation effects, the clinical application of ultrasound for transdermal delivery is still largely limited to the delivery of low-molecular-weight drugs, such as diclofenac and lidocaine (Becker et al., 2005, Liao et al., 2016).

To further increase the permeability of the ultrasound-treated skin to macromolecules (>500 Da), Kushner et al. (2004) used a chemical enhancer (sodium lauryl sulfate) to create a localized transport region (LTR) on the skin, where the permeability increased ∼80-fold and the electrical resistivity of the skin decreased ∼5000-fold. Mechanistic studies further indicated that the removal of intercellular lipids (∼30%) and the formation of transcellular pathways at the stratum corneum contribute to enhancing the permeability of the LTRs (Alvarez-Roman et al., 2003, Kushner et al., 2007). It is worth noting that the formation of LTRs has been used successfully to deliver macromolecules (fluorescein isothiocyanate-labeled oligonucleotides) and nanoparticles (20-nm quantum dots) into the skin (Paliwal et al., 2006, Tezel et al., 2004). Although these developments are encouraging, the clinical application of ultrasound and sodium lauryl sulfate for controlled transdermal delivery remains challenging because (i) the position and shape of the LTRs on the skin cannot be predicted, and (ii) the total area of the LTRs varies considerably, ranging from 10 to 40 mm².

One possible reason for the poor controllability of ultrasound-created LTRs is that previous studies used unfocused low-frequency ultrasound (e.g., 20 kHz) with large exposure areas (e.g., 1 cm²). We hypothesize that a highly focused, high-frequency ultrasound transducer with a well-refined exposure area will produce skin LTRs in a more controlled manner. We note that consistency in terms of the area of the skin LTRs has been improved through the use of higher-frequency ultrasound (256 kHz) for transdermal delivery (Schoellhammer et al. 2012). In this study, we used a 1.1 MHz focused ultrasound transducer to create a single-site LTR at the center of the delivery patch with a consistent circular shape and well-controlled delivery area. By controlling the ultrasound parameters to create skin LTRs with areas around 1 mm², we propose a new ultrasound transdermal delivery protocol, which we refer to hereafter as an ultrasound erosion method. Furthermore, we evaluated the permeability of the LTR by examining the diffusion depth of India ink and the total amount of fluorescence-labeled dextran molecules diffused. In addition, we investigated the immunization efficiency of our protocol through the transdermal delivery of hepatitis B surface antigen (HBsAg).