**3. Results**

**Figure 2** shows the PPIX formation after 3 h of cream incubation for human and porcine skin obtained by widefield fluorescence imaging. **Figure 3** shows the quantitative analysis of the PPIX formation by counting pixels of the images and spectroscopy collection acquired by fluorescence techniques. The result of this analysis was acquired by means of the average data collected from volunteers and animals. The results of the porcine skin model were also published previously [19]. The results in **Figure 3** show that the high amount of PPIX production in human and porcine skin occurs for all cream samples (ALA, M2, M3, M4, M5, M6, and MAL) after 3 h of cream application.

fluorescence techniques. From this study, it was possible to analyze which sample PPIX was produced quickest, and by means of a parameter called the index of fluorescence (IF50) it was possible to quantify the PPIX production in minutes. IF50 means 50% of maximum fluores-

**Figure 3.** Analysis of PPIX production in human and porcine skin models after 3 h of cream sample application evaluated

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Although **Figure 4** presents the results of the human skin study, **Table 1** shows the IF50 results for the study in human and porcine skin. We have included it here for best comparison. The graphs of the kinetics study in porcine can be observed in previously published work [19].

The results for the kinetics study in the human skin model show that PPIX production is faster with ALA than MAL and cream samples (M3, M4, M5, and M6) for both fluorescence analyses. However, the mixtures M3, M4, and M5 presented better results for PPIX production than MAL in the kinetics study (IF50 values). These results suggest that these differences may be due to high variability in human skin experiments. Perhaps these differences can be reduced

The results for the kinetics study from **Table 1** show that PPIX production in 5 h (IF50) in human skin models is faster for ALA than MAL, and the opposite occurs for porcine skin models where PPIX production is faster for MAL than ALA. This can be explained by the previous preparation for porcine skin where we can suggest that PPIX production by MAL can be optimized. The other sample creams (M3, M4, M5, and M6) show the same behavior

The values found for IF50 through the widefield fluorescence imaging data were closer to human and porcine skin models than the IF50 values collected by fluorescence spectroscopy. We believe that this occurred because PPIX production is heterogeneous and the fluorescence spectroscopy measurements are punctual. This punctual fluorescence collection data of PPIX production can suggest false negative or false positive results. On the other hand, by using widefield fluorescence imaging, we can evaluate all PPIX production on the skin surface.

cence value obtained for 5 h.

by (a) widefield fluorescence imaging and (B) spectroscopy fluorescence.

by using a number of human volunteers.

considering the standard deviation.

Fluorescence imaging shows that PPIX production is heterogeneous for healthy skin in both skin models. Even so, it is possible to verify the differences in PPIX formation to ALA, MAL, and mixtures from both. The results suggest that PPIX formation is greater for ALA than for MAL for both models. In addition, PPIX formation of all sample cream mixtures from ALA and MAL (M2, M3, M4, M5, and M6) was more elevated than MAL and is similar to ALA.

In addition, it is important to mention that for porcine skin preparation it was necessary to shave the back, and for human skin preparation the area was cleaned with physiological serum. This previous skin preparation can interfere with cream sample penetration on the skin as well as PPIX production. This explains the lowest PPIX production for all samples in human skin models when compared to porcine skin models (**Figure 3**).

**Figure 4** shows the kinetics study on human skin only, wherein the monitoring of PPIX production was carried out during 5 h, and the measurements were carried out hourly for both

**Figure 2.** Widefield fluorescence imaging after 3 h of cream sample application (ALA, M2, M3, M4, M5, M6, and MAL): (A) PPIX production in human skin (inner arm) and (B) PPIX production in porcine skin back.

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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

**Figure 2** shows the PPIX formation after 3 h of cream incubation for human and porcine skin obtained by widefield fluorescence imaging. **Figure 3** shows the quantitative analysis of the PPIX formation by counting pixels of the images and spectroscopy collection acquired by fluorescence techniques. The result of this analysis was acquired by means of the average data collected from volunteers and animals. The results of the porcine skin model were also published previously [19]. The results in **Figure 3** show that the high amount of PPIX production in human and porcine skin occurs for all cream samples (ALA, M2, M3, M4, M5, M6, and

Fluorescence imaging shows that PPIX production is heterogeneous for healthy skin in both skin models. Even so, it is possible to verify the differences in PPIX formation to ALA, MAL, and mixtures from both. The results suggest that PPIX formation is greater for ALA than for MAL for both models. In addition, PPIX formation of all sample cream mixtures from ALA and MAL (M2, M3, M4, M5, and M6) was more elevated than MAL and is similar to ALA.

In addition, it is important to mention that for porcine skin preparation it was necessary to shave the back, and for human skin preparation the area was cleaned with physiological serum. This previous skin preparation can interfere with cream sample penetration on the skin as well as PPIX production. This explains the lowest PPIX production for all samples in

**Figure 4** shows the kinetics study on human skin only, wherein the monitoring of PPIX production was carried out during 5 h, and the measurements were carried out hourly for both

**Figure 2.** Widefield fluorescence imaging after 3 h of cream sample application (ALA, M2, M3, M4, M5, M6, and MAL):

human skin models when compared to porcine skin models (**Figure 3**).

(A) PPIX production in human skin (inner arm) and (B) PPIX production in porcine skin back.

with an occlusive dressing.

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

MAL) after 3 h of cream application.

**3. Results**

**Figure 3.** Analysis of PPIX production in human and porcine skin models after 3 h of cream sample application evaluated by (a) widefield fluorescence imaging and (B) spectroscopy fluorescence.

fluorescence techniques. From this study, it was possible to analyze which sample PPIX was produced quickest, and by means of a parameter called the index of fluorescence (IF50) it was possible to quantify the PPIX production in minutes. IF50 means 50% of maximum fluorescence value obtained for 5 h.

Although **Figure 4** presents the results of the human skin study, **Table 1** shows the IF50 results for the study in human and porcine skin. We have included it here for best comparison. The graphs of the kinetics study in porcine can be observed in previously published work [19].

The results for the kinetics study in the human skin model show that PPIX production is faster with ALA than MAL and cream samples (M3, M4, M5, and M6) for both fluorescence analyses. However, the mixtures M3, M4, and M5 presented better results for PPIX production than MAL in the kinetics study (IF50 values). These results suggest that these differences may be due to high variability in human skin experiments. Perhaps these differences can be reduced by using a number of human volunteers.

The results for the kinetics study from **Table 1** show that PPIX production in 5 h (IF50) in human skin models is faster for ALA than MAL, and the opposite occurs for porcine skin models where PPIX production is faster for MAL than ALA. This can be explained by the previous preparation for porcine skin where we can suggest that PPIX production by MAL can be optimized. The other sample creams (M3, M4, M5, and M6) show the same behavior considering the standard deviation.

The values found for IF50 through the widefield fluorescence imaging data were closer to human and porcine skin models than the IF50 values collected by fluorescence spectroscopy. We believe that this occurred because PPIX production is heterogeneous and the fluorescence spectroscopy measurements are punctual. This punctual fluorescence collection data of PPIX production can suggest false negative or false positive results. On the other hand, by using widefield fluorescence imaging, we can evaluate all PPIX production on the skin surface.

**Figure 5** shows the correlation linear fitting to fluorescence measurements obtained through widefield fluorescence imaging and fluorescence spectroscopy. The fitting in **Figure 5** shows that the red fluorescence signal emitted by PPIX in the porcine and human skin was measured at 3 h following application of ALA and MAL cream mixtures. These results confirm that there is a correlation between both models since the equation line factor obtained was 0.9824,

**Table 1.** IF50 values for widefield fluorescence imaging and fluorescence spectroscopy collected over time (5 h) after

**Fluorescence spectroscopy**

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**IF50 (min)**

**Human skin Porcine skin Human skin Porcine skin**

ALA 103 ± 15 120 ± 10 234 ± 18 230 ± 7

M3 154 ± 18 40 ± 60 312 ± 6 114 ± 16 M4 127 ± 14 128 ± 8 225 ± 13 17 ± 20 M5 122 ± 21 97 ± 7 280 ± 12 131 ± 17 M6 207 ± 38 120 ± 20 260 ± 15 187 ± 25 MAL 233 ± 18 70 ± 5 388 ± 37 131 ± 9

M2 138 ± 27 134 ± 6 315 ± 13

sample cream application on human and porcine skin model surfaces.

The same linear fitting was performed for fluorescence collected through fluorescence spectroscopy (results not presented here). However, we do not find a correlation between both models (human and porcine skin) by this optical technique due to high variability during

**Figure 5.** The best correlation analysis between human and porcine skin models by widefield fluorescence imaging.

bordering on 1.0, the ideal linear fitting number.

fluorescence spectroscopy collection.

**Samples Widefield fluorescence imaging**

**IF50 (min)**

**Figure 4.** Kinetics of the PPIX production in human skin models by fluorescence spectroscopy and widefield fluorescence imaging evaluations for all cream samples (ALA, M2, M3, M4, M5, M6, and MAL).


**Table 1.** IF50 values for widefield fluorescence imaging and fluorescence spectroscopy collected over time (5 h) after sample cream application on human and porcine skin model surfaces.

**Figure 5** shows the correlation linear fitting to fluorescence measurements obtained through widefield fluorescence imaging and fluorescence spectroscopy. The fitting in **Figure 5** shows that the red fluorescence signal emitted by PPIX in the porcine and human skin was measured at 3 h following application of ALA and MAL cream mixtures. These results confirm that there is a correlation between both models since the equation line factor obtained was 0.9824, bordering on 1.0, the ideal linear fitting number.

The same linear fitting was performed for fluorescence collected through fluorescence spectroscopy (results not presented here). However, we do not find a correlation between both models (human and porcine skin) by this optical technique due to high variability during fluorescence spectroscopy collection.

**Figure 5.** The best correlation analysis between human and porcine skin models by widefield fluorescence imaging.

**Figure 4.** Kinetics of the PPIX production in human skin models by fluorescence spectroscopy and widefield fluorescence

imaging evaluations for all cream samples (ALA, M2, M3, M4, M5, M6, and MAL).

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

The fitting results shown in **Figure 5** indicate the best correlation between porcine and human skin models by widefield fluorescence imaging measurements. The possibility of predicting drug behavior on transdermal skin application promotes the success of clinical topical PDT treatment.

The kinetics study observed that ALA, M4, and M5 indicated the least time of PPIX production (high PPIX production velocity) at the skin. Both studies, human and porcine skin, showed the same behavior. IF50 values acquired by widefield fluorescence imaging for both models were very close, with the exception of M3, M6, and MAL. Thereby, it is possible to appreciate the similarity of porcine skin with human skin by first performing clinical tests

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However, it is known that *in vitro* and *in vivo* experiments using the same species show less variability than experiments using human volunteers. The authors suggest that human experiments are done using a greater number of volunteers. The measurement of the correlation coefficient proved that porcine and human skin models have the same behavior with respect to the production of PPIX in quantity as well as in speed of PPIX production through optical methods. The correlation coefficient is a measure of how well the predicted values from a forecast model fit with the real data. We suggest that the best correlation is between porcine and human skin by widefield fluorescence imaging, suggesting this optical method as an important tool to develop new clinical topical PDT protocols.

The correlation found between human and porcine skin models measured by widefield fluorescence imaging confirms that porcine skin can be used for establishing human protocols in clinical topical PDT using ALA, MAL, and mixtures from both. The capacity of porcine skin models to predict PDT results in humans can be beneficial to clinical studies optimizing PDT

The authors acknowledge the financial support of the National Council of Technological and Scientific Development (CNPq process 140370/2012-9) and São Paulo Research Foundation (FAPESP/CEPOF). Expressive thanks are extended to Ms. Michelle Requena and all the vol-

\*, Rozana Wendler da Rocha<sup>2</sup>

, Vanderlei Salvador Bagnato1

2 Department of Veterinary Clinic and Surgery, School of Agriculture and Veterinary

1 São Carlos Institute of Physics, University of São Paulo, São Carlos, SP, Brazil

, André Escobar2

and

,

on porcine skin.

**5. Conclusion**

treatment on patients.

**Acknowledgements**

**Author details**

Alessandra Keiko Lima Fujita1

Priscila Fernanda Campos de Menezes1

\*Address all correspondence to: alessandra.keiko@gmail.com

Sciences, São Paulo State University (UNESP), Jaboticabal, SP, Brazil

Andrigo Barboza de Nardi2

unteers for their participation in this study.
