1.

Introduction

The possibility of selective translucence of the superficial skin layers is very useful in developing functional imaging techniques, including optical coherence tomography (OCT) and reflectance spectroscopy. Recently, numerous works have been carried out in skin optical clearing, since it has a great potential in enhancing the capabilities of noninvasive light-based diagnostic and laser therapeutic techniques. Current challenges to skin optical clearing include epidermal penetration by optical clearing agents (OCA). The outermost layer of the skin, the stratum corneum (SC), presents a significant barrier to the most topically applied OCA and is hence responsible for the poor optical clearing effect. Slow diffusion of the index-matching agent through the skin barrier makes practical implementation of the approach difficult. To reduce barrier function of the skin during optical clearing, a number of different chemical and physical methods have been proposed. Chemical penetration enhancers, photothermal, sandpaper, and microneedle arrays have been shown to accelerate the skin permeability of OCA.^{1, 2, 3, 4, 5, 6} However, most of the current enhancing methods are invasive. Our recent studies have demonstrated that sonophoretic delivery (SP), as a noninvasive physical method, exhibited an enhancing the skin-clearing effect when applied topically with glycerol.⁷ The significant ultrasound-induced enhancement in OCT imaging depth and contrast of in vitro porcine skin and in vivo human skin was found.^{8, 9}

A vast number of studies have been reported on the sonophoretic delivery of small molecules through the skin in transdermal drug delivery (TDD).¹⁰ It represents a useful noninvasive physical method to enhance epidermal permeability. In spite of the wide use of sonophoretic delivery in TDD, there are few studies to develop sonophoretic delivery for optical clearing of skin. In this study, we further examine the effect of ultrasound on a series of alcohols, i.e., polyethylene glycol (PEG), glycerol, propylene glycol, butanediol, and butanol using in vitro porcine skin. The aim of the study is to evaluate the effects of various alcohols mediated with ultrasound on optical skin clearing and to find more effective ultrasound-OCA combinations for skin clearing.

2.

Materials and Methods

3.

Results and Discussion

Figures 1a and 1b show the examples of the shift of light transmittance spectra of the fresh porcine skin specimen with the topical application of 60% PEG200 (control) and 60% PEG200 with a $15\text{-}\min$ simultaneous application of ultrasound (PEG200/SP) over a range from $400\text{to}1400\mathrm{nm}$ , where the curves were obtained from the time intervals of 0, 15, 30, 45, and $60\min$ from bottom to top. Light transmittance increased against time for both treatments. The clearing capability of 60% PEG200 was much improved with the simultaneous application of ultrasound [Fig. 1b]. Other alcohol-ultrasound combinations exhibited similar effect with different levels (figures not shown). The dynamics of enhancement in transmittance at $1276\mathrm{nm}$ are summarized in Fig. 2 with (a) for the six alcohols alone and (b) for alcohol-ultrasound combinations at $60\min$ . For alcohols alone, glycerol exhibited the highest clearing effect $(\Delta T=37.8\%)$ and butanol had the lowest effect $(\Delta T=23.6\%)$ , consistent with previously reported results.^{11, 12} It is believed that smaller molecule with more hydroxyl groups in the chemical structure would penetrate the skin epidermis more easily, corresponding to the more enhanced transmittance. After application of alcohol-ultrasound combinations at $60\min$ , the total transmittance of the skin sample increased by 55.7%, 56.9%, 50.3%, 53.9%, 52.4%, and 31.2% for glycerol, PEG200, PEG400, propylene glycol, butanediol, and butanol, respectively [Fig. 2b]. The difference in clearing effect between alcohol alone and ultrasound-alcohol combination was significant $(P<0.05)$ for the six alcohols. Except for butanol, propylene glycol, PEG, and butanediol achieved almost the same enhanced effect as glycerol with ultrasound assistance. In addition, PEG200-ultrasound exhibited a 1.8-fold the enhanced effect of PEG200 alone, while glycerol-ultrasound did 1.5-fold of glycerol alone.

Fig. 1

Measured transmittance spectra over a range from $400\text{to}1400\mathrm{nm}$ for fresh skin treated with 60% PEG200 against time with intervals of 0, 15, 30, 45, and $60\min$ from bottom to top: (a) 60% PEG200, and (b) 60% PEG200 in implementation of $15\text{-}\min$ ultrasound.

Fig. 2

Enhancement in light transmittance at $1276\mathrm{nm}$ : (a) for alcohols alone, and (b) for alcohol-ultrasound combinations.

Increase of skin translucence can aid noninvasive aiming for diagnostic purposes. OCT was used as a noninvasive imaging modality. Figure 3 shows OCT images of the porcine skin treated with PEG200 alone and PEG200-ultrasound combination. It can be seen that light penetration is limited in native skin [see Figs. 3a and 3b, $0\min$ ], corresponding to a low imaging depth due to a shallow $1∕e$ light penetration depth of approximately $0.95\pm 0.05\mathrm{mm}$ [see Figs. 4a and 4b, lower lines]. A small enhancement with 60% PEG200 was found from Fig. 3a and Fig. 4a, upper line. The light penetration was greatly enhanced after $60\min$ of PEG200/SP exposure [see Fig. 3b]. Sixty minutes of PEG200/SP exposure resulted in a significantly increased $1∕e$ light penetration depth of $1.43\pm 0.03\mathrm{mm}$ [see Fig. 4b].

Fig. 3

OCT images of in vitro porcine skin at 0, 15, 30, and $60\min$ after topical application of (a) 60% PEG200 and (b) 60% PEG200 in implementation of $15\text{-}\min$ ultrasound.

Fig. 4

OCT intensity profiles of (a) native and 60% PEG200 and (b) native and 60% PEG200 in combination of ultrasound-treated in vitro porcine skin.

Figure 5 summarizes the relative $1∕e$ light penetration depth value for 60% Gly, Gly/SP, 60% PEG200, and PEG200/SP at $60\min$ . The depth increased by roughly 40%, 56%, 29%, and 56%, respectively. Consistent with the total transmittance data from spectroscopy measurement, results from OCT imaging that measures least scattering photons demonstrate that the PEG200-ultrasound combination is much more effective than PEG200 alone and exhibits the same effect as glycerol-ultrasound combination.

Fig. 5

Dynamic changes of light penetration depth into in vitro optically cleared porcine skin with 60% glycerol, Gly/SP, 60% PEG 200, and PEG200/SP at $1310\mathrm{nm}$ .

One of the major protective functions of the skin is derived from its uniquely low permeability. The excellent diffusion resistance of the SC makes the transdermal delivery of OCA and water loss through skin difficult. Subsequently, the optical clearing effect of intact skin is limited with topical application of OCA onto the surface of epidermis.¹³ Smaller substances $(<500\mathrm{Da})$ are used as OCAs for their ability to penetrate the skin more easily.¹⁴ In addition, if an OCA has more hydroxyl groups, it penetrates the skin more easily due to hydrophilic nature of the epidermis.¹⁴ Hence, without the aid of ultrasound, glycerol with smaller MW and three hydroxyl groups is more effective into the skin than PEG200 with two hydroxyl groups, as reported in Ref. 11 [Fig. 2a and Fig. 5a].

The constantly increasing interest in transdermal drug delivery has led to the development of innovative technologies in attempt to increase transdermal drug delivery. Among the noninvasive methods, ultrasound has demonstrated an enhancing transdermal mass transport effect when applied topically. Based on the preceding results, we hypothesize that, due to ultrasound penetration enhancing and convection effects, more alcohol can penetrate the skin and/or alcohol can penetrate more quickly to achieve more refractive index matching in the skin, therefore increasing light transmittance in the skin.^{7, 8, 9} Furthermore, sonophoretic delivery is effective in enhancing permeability for chemicals with MW from 76 to 400 and the enhanced effect is not dependent on solution osmolarity (Table 1).

With sonophoretic delivery, the optical clearing effect may not be directly related to the number of hydroxyl groups in the alcohol structure, or MW, or osmolarity. Although MW of PEG200 is almost twice that of glycerol, the osmolarity of 60% PEG200 is about twice less of 60% glycerol, PEG200 has one hydroxyl group less than glycerol (Table 1), and 60% PEG200 with the aid of ultrasound exhibits the same clearing effect as 60% glycerol-ultrasound. After the OCA is topically applied onto the surface of epidermis and allowed to diffuse into the dermis, the optical clearing effect probably depends on the quantity of an agent reaching the derma. Ultrasound is likely to drive same quantity of PEG200 and glycerol into the skin no matter the different osmolarity and MW between the two solutions. In addition, a similar effect was achieved by PEG200/SP as glycerol/SP only after $15\text{-}\min$ exposure. Obviously, ultrasound accelerates PEG200 penetration into the skin, as was reported for glycerol earlier.^{7, 8}

It should be noted that, in general, the stratum corneum in the fresh porcine skin obtain from an accredited abattoir may have been removed. Although we have already demonstrated the glycerol-ultrasound effect on in vivo human skin with stratum corneum in Ref. 8, if a skin sample with stratum corneum was used in this study, the results would be more pervasive and significant.

Polyethylene glycol is nontoxic, odorless, neutral, lubricating, nonvolatile, and nonirritating and is used in a variety of pharmaceuticals and in medications as a solvent, dispensing agent, ointment and suppository base, vehicle, and tablet excipient. PEG is characterized by less toxicity and less skin irritation.¹⁵ There is no damage in case of contact with skin or lips. It is the basis of many skin creams and sexual lubricants, frequently combined with glycerol. In addition, PEG200 and PEG400 have been used as penetration enhancers in transdermal drug delivery, and the effect is same as oleic acid for some drugs.¹⁶ Recently, the enhanced effect of penetration enhancers such as oleic acid,² azone,³ and sodium lauryl sulfate (SLS)⁹ on skin optical clearing have been demonstrated. We have reported the combined effect of ultrasound and the penetration enhancers with topical application of glycerol.^{7, 9} Hence, it can be expected that the coadministration of the mixture of PEG200 with glycerol under ultrasound-assistance may be a more effective approach in intact skin clearing as PEG200 can serve as both an optical clearing agent and penetration enhancers at the same time.

4.

Conclusions

It was demonstrated that simultaneous application of ultrasound and alcohols led to markedly increase in light transmission and imaging depth through in vitro porcine skin. Ultrasound-assisted enhancement was not related to the number of hydroxyl groups in the chemical structure and osmolarity of OCA solutions. PEG200 with ultrasound exhibited the highest enhancement among the six alcohols. It can be expected that an ultrasound-mediated mixture of glycerol and PEG200 should be a better solution for skin clearing based on the nature of PEG200 as a clearing agent and a penetration enhancer.