Sonophoresis or phonophoresis, defined as the ultrasonically facilitated delivery of drugs to or through the skin, has emerged as one of the most promising physical enhancers (Mitragotri 2013). In this technique, a piezoelectric crystal is responsible for converting electrical energy into acoustic waves. These waves are conducted from the sound source to the skin by a coupling medium (Herwadkar and Banga 2012). Ultrasound has been widely used in sports medicine since the 1950s. As shown in Table 26.2, ultrasound can be classified, according to the frequency range. In the case of the skin, transport enhancement has shown to be more efficient with low-frequency ultrasound (<100 kHz). Low-frequency high-intensity ultrasound waves (2–50 W/cm²) can form pores in the SC, allowing the transdermal delivery of macromolecules up to 48 kDa (Herwadkar and Banga 2012). In general, skin permeability increases with decreasing frequency and with increasing exposure time and intensity (Khafagy et al. 2007). Ultrasound can be applied continuously or in a sequential mode (pulsed mode). Continuous high-frequency ultrasound has shown to be less effective for transdermal drug delivery (Monti et al. 2001). Furthermore, temperature rises faster and more intense in the continuous mode (Machet and Boucaud 2002). Excellent reviews of sonophoresis have been published in the last years (Machet and Boucaud 2002; Lavon and Kost 2004; Mitragotri and Kost 2004; Haar 2007; Ogura et al. 2008; Polat et al. 2010; 2011b).

Sonophoresis was used for the first time in the 1950s to enhance the absorption of hydrocortisone. In general, low-frequency sonophoresis has shown to enhance the transdermal transport of drugs, reducing the associated lag time, being useful for the local and systemic delivery of drugs. Sonophoresis can be applied in two manners: (i) simultaneously with the drug and (ii) prior to drug delivery, pretreating the skin (Mitragotri and Kost 2004).

Within certain ultrasound intensity range, a direct dependence between intensity and permeability enhancement has been observed, i.e., enhancement increases from a threshold intensity until a maximum intensity value, beyond which, no further increase is observed. Besides ultrasound energy intensity, enhancement also depends on frequency, duty cycle, and application time. Due to the direct relationship between conductance and skin permeability, the effect of sonophoresis on skin permeabilization can be determined by conductance measurements. Ultrasound exerts a dose-dependent effect on skin permeability, with a range of safety: Low-frequency sonophoresis at low intensities (e.g., 2.5 W/cm²) appears to be safe and tolerable, producing minimal urticarial reactions, with no detectable changes in the structure of human skin. Therefore, suitable conditions must be carefully selected, in order to avoid undesirable effects. A low-frequency portable ultrasound device (SonoPrep Ultrasonic Skin Permeation System, Sontra Medical Corporation) for skin permeabilization has been approved by the US Food and Drug Administration (FDA) (Mitragotri 2004; Alexander et al. 2012).

As mentioned, important experimental variables include:

Low-frequency sonophoresis has shown to effectively enhance the transport of high molecular weight drugs, such as heparins, insulin, γ-interferon, and erythropoietin (Mitragotri et al. 1995; Mitragotri et al. 1996; Mitragotri and Kost 2004; Motlekar and Youan 2006; Herwadkar and Banga 2012). Apparently, cavitation (formation and immediate implosion of bubbles in a liquid) opens large pores and channels in the lipid layers of the SC, providing a less tortuous path for the transport of macromolecules. However, it is important to point out that in the case of the simultaneous application of ultrasound and the drug, the effect of ultrasound on protein conformation and/or activity should be determined (Khafagy et al. 2007).

Other applications of low-frequency ultrasound include (Mitragotri et al. 2000; Mitragotri and Kost 2004; Hosseinkhani and Tabata 2005; Frenkel 2008; Mitragotri 2013):

Apparently, transfollicular pathways are more susceptible to ultrasonic enhancement than are transcellular processes (Meidan et al. 1995). An important fact is that sonicated skin recovers its original barrier properties, with only slight histological changes. Some authors (Monti et al. 2001; Machet and Boucaud 2002) reported an immediate recovering of skin barrier once turning off sonication, while Lavon and Kost (2004) argued that a single short application (less than 2 min) of ultrasound may enhance skin permeability over several hours, recovering its normal value by 24 h. A greater enhancement activity of ultrasound has been found for hydrophilic molecules with respect to less hydrophilic ones (Monti et al. 2001).

In this section, studies related to the simultaneous use of sonophoresis and chemical enhancers are reviewed. Table 26.3 represents some of these results. However, it is important to mention that sonophoresis has also been combined with other physical or mechanical enhancement methods, such as microneedles (Chen et al. 2010; Han and Das 2013; Han and Das 2015; Pamornpathomkul et al. 2015) and iontophoresis (Ueda et al. 1996; Le et al. 2000; Fang et al. 2002; Watanabe et al. 2009). This kind of treatment has shown to greatly enhance the delivery rate of large molecular weight compounds into the skin. Furthermore, combination of US with some carriers has shown a greater permeation than when applied separately, e.g., polymeric, lipid-based nanoparticles, micelles and elastic liposomes (Husseini and Pitt 2008; Kasetvatin et al. 2015).

Chemical enhancers are known to permeabilize the skin by different mechanisms such as lipid fluidization, lipid extraction, or protein denaturation. It has been demonstrated that a combination of penetration enhancers showing different modes of action frequently exerts a synergistic effect on drug permeation (Mitragotri 2000). This is important, since it can enable a reduction in the dose/time of exposition to the enhancer in order to achieve the desired effect, reducing or even avoiding undesirable effects related to high concentrations of chemical enhancers. The synergistic effect of ultrasound and chemical enhancers is attributed to cavitation that may induce and promote the dispersion of chemical enhancers with SC lipids (Lee et al. 2010).

Johnson et al. (1996) showed that corticosterone flux was enhanced by a factor of 903 (skin permeability increase, 8.7) when the skin was treated with ethanol/linoleic acid (1:1) in combination with ultrasound. The same formulation enhanced the flux through the skin by a factor of 13,000 (skin permeability increase: 14.4), when combined with therapeutic ultrasound (1 MHz, 1.4 W/cm²). The authors attributed this effect to a lipid fluidization or to the formation of a separate bulk of lipid domain in the SC. A synergism was also observed for the simultaneous application of ultrasound and laurocapram (Azone^®). The authors suggested an enhancement in laurocapram diffusion into the skin due to ultrasonic thermal effects (Meidan et al. 1998). The combined application of oleic acid and low frequency ultrasound was shown to enhance the penetration of lanthanum nitrate (LaNO₃) into the viable epidermis through an intracellular pathway. LaNO₃ was found uniformly distributed both in the intracellular and intercellular domains (Lee et al. 2010). These authors also found a synergistic effect in LaNO₃ when ultrasound was applied on tape-stripped skin.

A significant synergistic effect has been reported for the combined use of low-frequency ultrasound and surfactants. Simultaneous application of ultrasound and sodium lauryl sulfate (SLS) significantly increases the permeability and conductivity of the skin (Lavon et al. 2005). In fact, addition of SLS to the coupling medium has shown a linear increase in skin permeability in an SLS concentration range of 0–1 % (Mitragotri et al. 2000). The concentration of 1 % SLS in a solution is approved by the FDA for many cosmetic and pharmaceutical products (Lavon et al. 2005). SonoPrep® (Sontra Medical Corporation) is an ultrasonic skin permeation system that enhances the effectiveness of topical anesthetics, shortening the onset of topical anesthetics action (Kim et al. 2012). This system operates with 1 % SLS solution, showing no skin irritation (Boucaud 2004; Lavon et al. 2005).

Mitragotri et al. (2000) found a dramatic increase in mannitol permeability (200-fold enhancement) when 1 % SLS and ultrasound are combined. The authors also reported a reduction of the threshold ultrasound energy to produce a change in skin impedance, from 141 J/cm² (without SLS) to 18 J/cm² (with 1 % SLS). A reduction of treatment times, higher connectivity of lacunar regions of the SC, as well as a lower perturbation of the skin have been also demonstrated when SLS is used with sonophoresis, compared to sonophoresis alone (Polat et al. 2012). The synergistic enhancement of ultrasound and SLS was clearly demonstrated by Lavon et al. (2005), who found an enhancement ratio of ~ 35, while when applied separately, enhancement ratios were 4 for SLS and 12 for ultrasound. Polat et al. (2011a) studied the permeability of calcein at three frequencies (20, 40, and 60 kHz), with important contribution to the following points:

In order to investigate the synergism between ultrasound and SLS, Polat et al. (2011c) utilized two skin models, pig full-thickness skin (FTS) and pig split-thickness skin (STS), with two kinds of treatments, ultrasound (US) alone and ultrasound with SLS (US/SLS). Samples were treated with ultrasound until they reached currents between 5 and 200 μA. The authors studied the treatment time required to attain specific levels of skin electrical resistivity and found that US/SLS treatment reduced dramatically the time necessary to reach similar skin electrical resistivity values, with respect to the US-treated samples. Furthermore, the presence of SLS induced a more reproducible skin perturbation.

Sodium dodecyl sulfate (SDS) at 1 % (w/v) has also been used as the coupling medium, to investigate the influence of liposomes on in vitro antigen permeation and on in vivo skin barrier properties, using rats as animal models. The authors found important skin damage attributed to the synergistic effect of ultrasound and SDS (Dahlan et al. 2009). Similar findings were previously reported by Mitragotri et al. (2000) and Tezel et al. (2002).

Physicochemical characteristics of surfactants determine their action. Apparently, ionic surfactants are better enhancers than nonionic surfactants, and 14-carbon tail length in the surfactant molecule is the optimum to attain a synergistic effect with low-frequency ultrasound (Tezel et al. 2002).

Surfactant–sonophoresis synergism can be attributed to:

The physical phenomena involved in the combined use of sonophoresis and permeation enhancers have been well explained (Lee et al. 2010; Polat et al. 2012; Azagury et al. 2015). The mechanisms are related to the following aspects:

It is interesting to mention that in some cases, a greater enhancement activity has been reported for chemical enhancers alone than for low-frequency ultrasound alone. In this sense, Monti et al. (2001) found a higher enhancement effect on caffeine permeation with oleic acid/propylene glycol than with low-frequency ultrasound.

Electroporation was originally used to transfect cells with macromolecules such as DNA by altering their cell membranes, afterward it has been applied in tissues (e.g., for gene therapy) to reversibly permeabilize them (Denet et al. 2004). The investigation of electroporation for drug delivery began in the 1990s (Banga 2011). In recent years, electroporation has been proposed for delivery of DNA vaccines and for electrochemotherapy, which consists of applying high-voltage pulses to permeabilize tumor cells against cytotoxic drugs (Denet et al. 2004).

Electroporation or electropermeabilization is an electric enhancement technique that involves application of high-voltage pulses (50 to 1500 V) for very short durations of time (micro- to milliseconds) to induce the transitory structural perturbation of lipid bilayer membranes (Higaki et al. 2003; Brown et al. 2006; Banga 2011; Alexander 2012; Wong 2014). These intense electric charges generate structural rearrangements of the cell membrane and electrical conductance and changes of membrane conductance leading to a temporary loss of semipermeability of cell membranes (Banga 2011). The electroporation of the lipid bilayers of the SC causes the formation of transient aqueous pathways or electropores through it. These electropores allow drugs to penetrate more readily (Denet et al. 2004; Wong 2014; Ita 2015).

The electroporation mechanism has been comprehensively reviewed by several authors (Weaver and Chizmadzhev 1996; Weaver et al. 1999; Pliquett 1999; Sen et al. 2002a; Denet et al. 2004; Becker et al. 2014). The most important factors in the enhancement of percutaneous absorption of drugs by electroporation are the selection of the electrical parameters: pulse waveform (exponential decay or square wave), pulse voltage, pulse length, number of pulses, and spacing between pulses ). (Denet et al. 2004; Banga 2011; Zorec et al. 2015). Usually, the skin transport of the solutes improves when the applied voltages are increased (Higaki et al. 2003). The application of high-voltage electric pulses may be accompanied by temperature changes in the pulsing medium. A long-duration pulse could increase skin irritation (Banga 2011). The transdermal voltage is only a fraction of the voltage applied across the electrodes (ca. 10–50 %) since a significant voltage drop occurs within the electroporation system (Denet et al. 2004).

Furthermore, physicochemical properties of the drug and drug concentration can affect the transdermal drug delivery by electroporation (Vanbever and Préat 2000). The pK_a of the drug and the pH of the delivery solution influence the electric charges of the molecule to be delivered. Neutral molecules can diffuse through the permeabilized skin (passive diffusion); however, their transport is lower than the transport of charged molecules during pulses (Denet et al. 2004). Electroporation has been successfully used to enhance the skin permeability of molecules with different lipophilicity and size, including biopharmaceuticals with a molecular weight greater than 7 kDa, the current limit for iontophoresis (Mathur et al. 2010). Small- and moderate-sized molecules such as fentanyl, timolol, metoprolol, mannitol, calcein and cyclosporine A, etc., have been transported transdermally by electroporation (Denet et al. 2004). The influence of the molecular weight of the permeant on transdermal and topical transport by skin electroporation was first evaluated by Lombry and coworkers (Lombry et al. 2000). Fluorescein isothiocyanate (FITC) and fluorescein isothiocyanate–dextran (FD) macromolecules with increasing molecular weight (4.4, 12, and 38 kDa) were used as fluorescent permeant models. The transport of FITC and FD across hairless rat skin in vitro and their distribution in the skin was explored by confocal scanning laser microscopy. The results showed that it was feasible to deliver large macromolecules (at least up to 40 kDa) across the skin by electroporation. Concurrently, authors demonstrated that the macromolecule dextran enhanced FITC transport.