**3.2.1 Materials and methods**

The PA setup employed in such *in vivo* skin measurements consisted of a 250W tungsten halogen lamp as light source (with wavelength range 400nm<λ<700nm and light intensity of about 20W/m2), a mechanical chopper (SRS, model SR540), a lock-in amplifier (SRS, model SR530) and a microcomputer for data acquisition.

Fig. 2. Experimental setup with volunteer positioned for *in vivo* skin measurement

The double faced PA cell employed, with an electret microphone, was developed at UNIVAP; sensitivity was 15 mV/Pa for the frequency employed in skin measurements, 17Hz. The electret microphone structure was described by Marquezini et al*.* (1990). The PA cell has a cylindrical body and two opposite, parallel faces (one is closed by a thin glass layer and the other, by the sample itself). For the modulation frequency employed (17Hz), the thickness of the skin layer under study is about 30µm.

PA measurements were recorded as a function of time (200 readings for each measurement, in 0.5s intervals, up to a total of 100s per measurement). During measurements, one face of the PA cell was closed with a thin transparent window, while the forearm of the volunteer was gently pressed against the opposite face.

Figure 2 shows the PA experimental setup employed for in vivo skin measurements at the FASBio/UNIVAP, with a volunteer positioned for measurement.

Measurements were performed in 57 female volunteers, between 20 and 30 years-old. Initially, each volunteer answered a questionnaire according to their daily routine associated to skin care; volunteers were also clinically evaluated and, as a result, they were classified according to skin phototype, following Fitzpatrick classification (Fitzpatrick, 1988).

Before measurements, the skin area to be evaluated was cleaned with cotton embedded in alcohol 70%. The PA signal was then recorded for the inner and outer faces of both forearms. Volunteers were then classified according to the respective PA signal amplitude, and this classification was compared to the phototype classification.

## **3.2.2 Results and discussion**

292 Acoustic Waves – From Microdevices to Helioseismology

were tested using the PA probe and visible reflectance spectroscopy (VRS); melanin content was evaluated through each of these methods, and a good linear fit (r2=0.85) was obtained

Pigmentation skin level can also be evaluated through simple, direct PA measurements employing non-laser light sources. Actually, PA measurements have been performed at the Laboratory of Photoacoustic Technique Applied to Biological Systems (FASBio), at UNIVAP (Brazil). The objective of such *in vivo* measurements was to classify different skin types according to the amplitude of the PA signal, which can be associated to the corresponding

In the following subsections, we present this straightforward PA approach to skin characterization according to the level of pigmentation, employing PA measurements in volunteers. Experimental results are compared both to Fitzpatrick and Baumann clinical

The PA setup employed in such *in vivo* skin measurements consisted of a 250W tungsten halogen lamp as light source (with wavelength range 400nm<λ<700nm and light intensity of about 20W/m2), a mechanical chopper (SRS, model SR540), a lock-in amplifier (SRS, model

Fig. 2. Experimental setup with volunteer positioned for *in vivo* skin measurement

The double faced PA cell employed, with an electret microphone, was developed at UNIVAP; sensitivity was 15 mV/Pa for the frequency employed in skin measurements,

for the plot of PA x VRS.

pigmentation level of the skin.

**3.2.1 Materials and methods** 

SR530) and a microcomputer for data acquisition.

skin type evaluations.

Initially, a comparison between the PA signal amplitude of the inner and outer faces was performed, showing a highly significant statistical difference (paired t-test, p<0,005), with higher PA amplitude being observed for the outer face of the forearm. This result can be attributed to the higher pigmentation level of the skin region continuously exposed to solar radiation, demonstrating that skin constitution and aspect are clearly influenced by the level of sun exposure.

After clinical evaluation of the volunteers for skin phototype (following Fitzpatrick classification), PA results were grouped according to the phototype of each volunteer. Results are presented in Table 1.


Table 1. PA signal amplitude (mV) for the inner face of the forearm, for each skin phototype (average ± standard error). Different indexes (a, b) indicate significant statistical difference (comparison among groups: p=0.009, ANOVA)

Comparison among phototype groups was performed and significant statistical difference was verified (as we can see in Table 1), showing that the PA signal level (amplitude) for the inner forearm tends to scale with skin phototype, as defined by Fitzpatrick.

Afterwards, the PA signal amplitude for each volunteer (average values for the inner face of the forearm) allowed the division of the volunteers in two groups, "pigmented" (P) and "non-pigmented" (NP), following the Baumann proposal. As the average PA signal

Photoacoustic Technique Applied to Skin Research:

can be sucessfully employed in skin permeatin studies.

**4.3 Photoacoustic evaluation of topically applied products** 

(for nitrofurazona, vaseline and vaporub) to the experimental data.

longer protection against UV radiation).

human skin.

permeation.

product).

Characterization of Tissue, Topically Applied Products and Transdermal Drug Delivery 295

rabbit and pig skin are, by far, the most employed alternatives, because of the similarity to

As pointed by Simon & Malbach (2000), physiological and anatomical similarities between man and pig make this animal a good model for man in biomedical research. The correlation of quantitative data between pig skin and human skin can be frequently classified as very good (Benech-Kieffer et al., 2000); therefore, pharmacological (and even toxicological) skin research is often based on the knowledge of pig skin absorption and percutaneous

Recently, Nicoli et al*.* (2008) employed qualitative and quantitative analysis of stratum corneum lipids and permeation experiments to analyze the utilization of rabbit ear skin in transdermal permeation studies, using pig ear skin as a reference. Their results showed that the stratum corneum of both rabbit ear skin and pig ear skin present similar thickness. Probably due to its higher lipophilicity, rabbit ear skin was less permeable to hydrophilic compounds; however, the permeability to progesterone was comparable between isolated pig epidermis and rabbit ear skin. Nicoli and co-workers conclude that the rabbit ear skin

Different pharmaceutical formulations for a topically applied drug may present very different transdermal delivery ratios, depending on the product composition (excipients usually play a major role in the penetration kinetics of topically applied products). These penetration rates may be evaluated through the analysis of the time-dependence of the PA signal after topical application of a given product in skin. This methodology can also be applied to the evaluation of sunscreens, that may be characterized in terms of their (photo)stability after topical application (in this case, the lower the rate, the better the

Gutierrez-Juarez et al. (2002) employed PA measurements in the analysis of substances topically applied to the human skin. To fulfill this purpose, these authors utilized a doublechamber PA cell; the absorption determination was obtained through the measurement of the thermal effusivity of the binary system substance–skin. The model employed by Gutierrez-Juarez and co-workers (that assumes that the effective thermal effusivity of the binary system corresponds to that of a two-phase system) was experimentally applied to study different topically applied substances, in different parts of the body. The corresponding relative concentrations of substances as a function of time were determined by fitting a sigmoidal function (for ketoconazol and sunscreen) or an exponential function

Pedrochi and co-workers (2005) employed PAS measurements to evaluate the penetration rate of different sunscreens into human skin *in vivo*. Their results showed that the diminution rate of the sunscreen amount in the skin surface depends on the form of the product: sunscreens in cream form tend to present faster reduction after application in skin. This leads to the conclusion that sunscreens in gel form are more adequate (presenting

Another transdermal drug delivery study is the work of Truite et al*.* (2007), which employed PAS measurements in the *ex vivo* determination of the penetration rate of different phytotherapic formulations (with and without salicylic acid) for treatment of vitiligo. Measurements were performed as a function of time in rabbits. PA depth monitoring

amplitude obtained for all measurements (inner face of the forearm) was 1.5mV, this was the cutoff value adopted for separating the volunteers into "P" (for PA signal amplitude above 1.5 mV) and "NP" (under 1.5 mV). Table 2 shows the division of each (clinically evaluated) phototype group into the (experimentally evaluated) P and NP groups. In this way, the PA technique allowed the comparison between two different skin classification forms.


Table 2. Distribution of the volunteers of each phototype in the NP and P groups, according to the PA signal level

Table 2 shows that phototype II is highly related to the NP group, while phototypes IV and V concentrate in the P group. Phototype III appears in both groups, showing the variability of elements inside this classification.

The simple methodology presented here and the corresponding results obtained open perspectives for an objective, experimental classification of skin types, based upon PA measurements. Additional work in this field is currently being performed at FASBio/UNIVAP (Brazil).
