**2. Materials and methods**

#### **2.1. Chemicals**

The PPIX precursors used in this study were ALA and MAL (final concentration of 20%), which were dissolved in different proportions in an oil-in-water (O/W) emulsion. Seven samples (ALA, MAL, and mixtures from both) were prepared in the following proportions: M1 (100% ALA), M2 (80% ALA and 20% MAL), M3 (60% ALA and 40% MAL), M4 (50% ALA and 50% MAL), M5 (40% ALA and 60% MAL), M6 (20% ALA and 80% MAL), and M7 (100% MAL). The emulsion or cream preparation was previously described [19].

The areas were dressed with an occlusive mask to protect them from light. The cream was removed prior to autofluorescence collection at the skin by fluorescence spectroscopy and widefield fluorescence imaging. After collection, the same amount of cream was applied again under the same conditions at every full hour during 5 h. The experimental procedure

Correlation between Porcine and Human Skin Models by Optical Methods

http://dx.doi.org/10.5772/intechopen.75788

165

A widefield fluorescence imaging system was used for image acquisition. It is a commercial device, produced by MM Optics, Sao Carlos, Brazil, called EVINCE. In brief, the widefield imaging system consists of a lighting device based on LEDs, emitting around 405 nm coupled to a digital camera for image acquisition [2, 19, 20]. The measurements were taken for each sample and different times of PPIX formation. The images obtained by widefield fluorescence were assessed quantitatively using a routine written in Matlab program. The program has

For pixel count analysis, standardization was obtained dividing the red channel by the green channel. This was done to avoid the effects of different shutter speeds and ISO settings for image acquisition. Finally, fluorescence (pixel count) was determined by summing the values of all red channels divided by the sum of all values of the green channel. With these data, it was possible to quantify PPIX production (which shows red fluorescence) in amount and

For fluorescence spectroscopy analysis, a system with two excitation lasers was used, emitting at 408 nm and 532 nm, respectively. The investigation fiber probe in the Y shape directs the excitation laser to the tissue, while the second arm of the Y shape collects the re-emitted light from the tissue and delivers light to a spectrophotometer. The spectrophotometer used was the USB2000 (Ocean Optics®, USA). A filter was used to remove backscattered light so that

We recorded the fluorescence spectrum in contact with the tissue at five points in the area where the cream was applied, and equivalent autofluorescence collection was performed. The evaluations from spectral analysis were normalized by total area and subtracted from

**Figure 1.** Application of different cream samples (ALA, M2, M3, M4, M5, M6, and MAL) at the skin: (A) human right

**2.4. Fluorescence collection: widefield fluorescence imaging and fluorescence** 

defined an array separating the red, green, and blue (RGB) channel colors.

was previously described (**Figure 1**) [2, 19].

uniformity on the skin extension [2, 19].

only the fluorescence was collected [2, 19, 21].

inner arm; (B) human left inner arm; and (C) porcine skin back.

**spectroscopy**

Commercial ALA and MAL were obtained from PDT-PHARMA (Cravinhos, São Paulo, Brazil) and were prepared immediately prior to use without previous solubilization because the drug presents elevated solubility in the base cream used.

## **2.2. Human study approval**

This study used a protocol along the lines of the procedures established by Brazil's Human Research Ethics Committee (no. 13556713.8.0000.5504). In addition, a written informed consent was obtained from all participants. Ten female patients aged around 25 years with the clinical diagnosis of normal skin were recruited for this study. To be considered eligible, a patient had to be free of skin disorders on both arms. The volunteers were all women to decrease the variable numbers in the study.

All patients had either Fitzpatrick skin type II (50%) or III (50%). Patients with lesions on the target area or with porphyrin were excluded. Additional exclusion criteria included male volunteers, pregnant or lactating female volunteers, those allergic to ALA and MAL, and volunteers less than 20 and more than 35 years of age. The volunteer protocol followed the standardization previously described [2].

#### **2.3. Topical application of the skin models**

The study was performed *in vivo* in human and porcine skin models using ALA, MAL, and mixtures from both cream applications (samples: ALA, M2, M3, M4, M5, M6, and MAL). The cream was applied with a sterile spatula, with a density of 32 mg/cm2 , in tests involving both animals and human volunteers.

In human skin, the cream was applied in seven areas (a circular area of 1 cm2 each) on each volunteer's right or left inner arm (10 woman human volunteers). Before cream application, the area was cleaned with serum. Skin autofluorescence was used as the control for each cream sample (ALA, M2, M3, M4, M5, M6, and MAL). Data were compared for each treatment group for the same volunteer and among volunteers.

In porcine skin, the cream was applied in a square area of 4 cm<sup>2</sup> (applied on the back). To prepare the cream application it was necessary to remove the hairs from the back. Animal testing involving porcine skin models was performed as described above with seven cream samples (ALA, M2, M3, M4, M5, M6, and MAL) in triplicate (in regard to conditions as well as experiments) [19].

The areas were dressed with an occlusive mask to protect them from light. The cream was removed prior to autofluorescence collection at the skin by fluorescence spectroscopy and widefield fluorescence imaging. After collection, the same amount of cream was applied again under the same conditions at every full hour during 5 h. The experimental procedure was previously described (**Figure 1**) [2, 19].

#### **2.4. Fluorescence collection: widefield fluorescence imaging and fluorescence spectroscopy**

**2. Materials and methods**

164 Human Skin Cancers - Pathways, Mechanisms, Targets and Treatments

**2.2. Human study approval**

decrease the variable numbers in the study.

standardization previously described [2].

animals and human volunteers.

as experiments) [19].

**2.3. Topical application of the skin models**

The PPIX precursors used in this study were ALA and MAL (final concentration of 20%), which were dissolved in different proportions in an oil-in-water (O/W) emulsion. Seven samples (ALA, MAL, and mixtures from both) were prepared in the following proportions: M1 (100% ALA), M2 (80% ALA and 20% MAL), M3 (60% ALA and 40% MAL), M4 (50% ALA and 50% MAL), M5 (40% ALA and 60% MAL), M6 (20% ALA and 80% MAL), and M7 (100%

Commercial ALA and MAL were obtained from PDT-PHARMA (Cravinhos, São Paulo, Brazil) and were prepared immediately prior to use without previous solubilization because

This study used a protocol along the lines of the procedures established by Brazil's Human Research Ethics Committee (no. 13556713.8.0000.5504). In addition, a written informed consent was obtained from all participants. Ten female patients aged around 25 years with the clinical diagnosis of normal skin were recruited for this study. To be considered eligible, a patient had to be free of skin disorders on both arms. The volunteers were all women to

All patients had either Fitzpatrick skin type II (50%) or III (50%). Patients with lesions on the target area or with porphyrin were excluded. Additional exclusion criteria included male volunteers, pregnant or lactating female volunteers, those allergic to ALA and MAL, and volunteers less than 20 and more than 35 years of age. The volunteer protocol followed the

The study was performed *in vivo* in human and porcine skin models using ALA, MAL, and mixtures from both cream applications (samples: ALA, M2, M3, M4, M5, M6, and MAL). The

volunteer's right or left inner arm (10 woman human volunteers). Before cream application, the area was cleaned with serum. Skin autofluorescence was used as the control for each cream sample (ALA, M2, M3, M4, M5, M6, and MAL). Data were compared for each treat-

prepare the cream application it was necessary to remove the hairs from the back. Animal testing involving porcine skin models was performed as described above with seven cream samples (ALA, M2, M3, M4, M5, M6, and MAL) in triplicate (in regard to conditions as well

, in tests involving both

(applied on the back). To

each) on each

cream was applied with a sterile spatula, with a density of 32 mg/cm2

ment group for the same volunteer and among volunteers.

In porcine skin, the cream was applied in a square area of 4 cm<sup>2</sup>

In human skin, the cream was applied in seven areas (a circular area of 1 cm2

MAL). The emulsion or cream preparation was previously described [19].

the drug presents elevated solubility in the base cream used.

**2.1. Chemicals**

A widefield fluorescence imaging system was used for image acquisition. It is a commercial device, produced by MM Optics, Sao Carlos, Brazil, called EVINCE. In brief, the widefield imaging system consists of a lighting device based on LEDs, emitting around 405 nm coupled to a digital camera for image acquisition [2, 19, 20]. The measurements were taken for each sample and different times of PPIX formation. The images obtained by widefield fluorescence were assessed quantitatively using a routine written in Matlab program. The program has defined an array separating the red, green, and blue (RGB) channel colors.

For pixel count analysis, standardization was obtained dividing the red channel by the green channel. This was done to avoid the effects of different shutter speeds and ISO settings for image acquisition. Finally, fluorescence (pixel count) was determined by summing the values of all red channels divided by the sum of all values of the green channel. With these data, it was possible to quantify PPIX production (which shows red fluorescence) in amount and uniformity on the skin extension [2, 19].

For fluorescence spectroscopy analysis, a system with two excitation lasers was used, emitting at 408 nm and 532 nm, respectively. The investigation fiber probe in the Y shape directs the excitation laser to the tissue, while the second arm of the Y shape collects the re-emitted light from the tissue and delivers light to a spectrophotometer. The spectrophotometer used was the USB2000 (Ocean Optics®, USA). A filter was used to remove backscattered light so that only the fluorescence was collected [2, 19, 21].

We recorded the fluorescence spectrum in contact with the tissue at five points in the area where the cream was applied, and equivalent autofluorescence collection was performed. The evaluations from spectral analysis were normalized by total area and subtracted from

**Figure 1.** Application of different cream samples (ALA, M2, M3, M4, M5, M6, and MAL) at the skin: (A) human right inner arm; (B) human left inner arm; and (C) porcine skin back.

the autofluorescence. The spectrum evaluations were performed using an Origin 9 program as previously described in our publication [2, 19]. A spectrometer and widefield fluorescence imaging equipment collected skin fluorescence at every full hour during 5 h. At the end of the fluorescence analysis, the cream mixtures were reapplied to the treatment area and covered with an occlusive dressing.
