Photomedicine and Laser Surgery Volume 32, Number 5, 2014 ª Mary Ann Liebert, Inc. Pp. 296–301 DOI: 10.1089/pho.2013.3670
Optimization of Photodynamic Therapy Using Negative Pressure Priscila Fernanda Campos Menezes, PhD, Michelle Barreto Requena, BS, and Vanderlei Salvador Bagnato, PhD
Abstract
Objective: The goal of this study is to demonstrate an alternative procedure to perform topical photodynamic therapy (PDT). Here, we propose the combined use of negative pressure and a 5-Aminolevulinic acid (5-ALA) cream occlusion to increase protoporphyrin IX (PPIX) formation. Background data: PDT using topical 5-ALA as a prodrug and precursor of PPIX has been used in the treatment and diagnosis of different types of cancer and skin diseases. The use of 5-ALA offers many advantages as a localized and non-systemic application, but it shows limitations in relation to skin penetration. Many authors have discussed the limitations of 5-ALA penetration through the skin. The skin penetration of 5-ALA can be optimized using mechanical devices associated with typical PDT procedure. Methods: For this study, 20% 5-ALA cream was applied to a 9 cm2 area of skin, and an occlusive dressing was placed. The PPIX production was collected at the skin surface, using fluorescence spectroscopy and widefield fluorescence imaging, for 7 h, and after 24 h. Results: We observed that in the presence of negative pressure therapy, the PPIX production, distribution, and elimination are greater and faster than in the control group. The PPIX formation was *30% in deeper skin layers, quantified by fluorescence spectroscopy analysis, and *20% in surface skin layers, quantified by widefield fluorescence imaging analysis. Conclusions: Negative pressure induction can also help PDT application in the case of inefficient PPIX production. These results can be useful for optimizing the PDT. Introduction
P
hotodynamic therapy (PDT) is a procedure that exploits the consequences originating from localized oxidative damage inflicted by photochemical processes.1 There are three critical elements required for PDT: a drug that can be activated by light [a photosensitizer (PS)], light, and oxygen. Light interacting with a PS produces an excited triplet state, which interacts with ground state oxygen creating singlet oxygen (102).1–3 The highly reactive singlet oxygen promotes oxidative damage in the immediate vicinity of the subcellular site of a localized PS.3 The tissue response to PDT depends upon adequate tumor oxygenation, as well as sufficient accumulation of a PS.4 PDT, using 5-Aminolevulinic acid (5-ALA), as a precursor of protoporphyrin IX (PPIX), has been used in the treatment and diagnosis of different types of cancer, skin diseases, and skin aging.1,5 5-ALA is found naturally in our body, and is converted into PPIX, the immediate precursor of heme. Because of the feedback mechanism, the excess exogenous 5-ALA results in intracellular accumulation of PPIX. PPIX is the endogenous PS used in topical PDT.6
5-ALA can permeate easily into the epidermis of cancerous tissue, because of altered metabolism, but not normal epidermis, thus creating highly selective photosensitivity in tumor tissue.1 All topical application interference involving 5-ALA during PDT can be summarized accordingly: problems related to chemical and physicochemical characteristics of 5-ALA, problems with the light system (ideal fluence), tumor surface characteristics (tumor depth and skin thickness), adjuvant mechanical techniques used in the treatment, and increasing the temperature of the skin.7 Therefore, 5-ALA penetration through skin depends upon the optimization of all these parameters.6–8 Another important aspect, normally not considered, is the condition of tissue in relation to its ability to convert 5- ALA in PPIX. Negative pressure therapy, also known as vacuum therapy, has been used to heal wounds.9,10 The negative pressure affects the microvascular blood flow by increasing the blood flow. This alteration in the blood flow occurs because of modification in the vascular diameter, blood flow velocity, blood volume, and others things.10,11 The purpose of this work is to show that it is possible to increase the formation, distribution, and elimination of PPIX, produced by its precursor 5-ALA through the skin,
Instituto de Fı´sica de Sa˜o Carlos (IFSC), University of Sa˜o Paulo (USP), Sa˜o Carlos - SP, Brazil.
296
5-ALA, PHOTODYNAMIC THERAPY
using negative pressure. The possibility of increasing the amount of PPIX production, decreasing the interval between the application of 5-ALA cream, and irradiation time, can be useful to clinical PDT in transdermal applications. In conclusion, the negative pressure can also help PDT application in the case of inefficient PPIX production, as it grows the production and uniformity of PPIX in the skin, increasing the effectiveness of PDT. Materials and Methods Chemicals
The studies were done using 5-Aminolevulinic acid hydrochloride (5-ALA) with a 20% concentration, in an oilin-water emulsion (O/W) obtained from PDT-PHARMA (Brazil). The formulation was evenly applied to a circular area of 9 cm2 with a sterile spatula, using a density of 32 mg/cm2. The area was dressed with an occlusive mask to protect it from light. Equipment for negative pressure induction
The vacuum prototype used to stimulate PPIX production in the skin can be adjusted from 0 to 550 mm Hg (negative pressure), operating in continuous or pulsed mode. This study was performed using 200 mm Hg for 4 min, during which we alternated between continuous mode (5 sec) and pulsed mode (55 sec). The temperature increases in the skin were evaluated using an infrared thermometer (ST-600, Incoterm, Sa˜o Paulo) at 1 h intervals. Volunteers: inclusion and exclusion criteria
The protocol used in this study followed the procedures establish by Human Research Ethics Committee of Brazil. Written informed consent was obtained from all participants. Ten women patients, *25 years of age with a clinical diagnosis of normal skin, were recruited for this clinical study. All patients had Fitzpatrick skin types II (50%) or III (50%). To be considered eligible, a patient had to be free of skin disorders on both arms. Patients with lesions in the target area or with porphyria were excluded. Additional exclusion criteria included pregnancy, lactation, an allergy to 5-ALA, male gender, and being < 20 or > 35 years of age. Treatment procedure
The study was randomized with two treatment groups. The first treatment group was treated with 20% 5-ALA cream and a vacuum device for negative pressure induction (NP-G). For the second treatment group, the control group (CG), treatment was performed on the volunteers’ other arm. Autofluorescence of the skin was used as the control for each group. Comparisons were made for each treatment group and between both treatments. First, the 5-ALA cream and the vacuum device were applied for 4 min. Following this, the fluorescence was measured at the skin surface using a spectrometer, and pictures of the area were taken using widefield fluorescence imaging equipment, as previously described. At the end of the fluorescence analysis, 5-ALA cream was reapplied to the treatment area and covered with an occlusive dressing. The same procedure was performed on the other arm without the use of the vacuum device.
297 Fluorescence spectroscopy
Fluorescence collection was done using a system composed of a doubled Nd:YAG laser emitting at 532 nm, and a spectrometer, collecting fluorescence in the range of 400– 1000 nm as previously described by us.12 For fluorescence spectrum analysis, we collected the fluorescence spectrum in contact with the tissue at five points in the 9 cm2 circular area. At each point, 20 fluorescence and autofluorescence measurements were performed. The evaluations from spectra analyses were performed using the maximum value of the fluorescence spectrum (at 633 nm) from PPIX for 7 h. The spectrum evaluations were performed using Matlab 12 platform (The MathWorks, USA). Widefield fluorescence imaging
For this study, commercial equipment called ‘‘Evince’’ was used (MM Optics, Brazil), which is composed of a lighting device based on LEDs, emitting *400 and 450 nm, a high-pass filter, dichroic mirrors, a band pass filter, and a CCD camera for image acquisition. The widefield fluorescence imaging was collected at the skin surface at each hour, from 1 to 7 h, and in 24 h. The analysis was performed by collecting five images of the treatment area each hour and evaluating the pixel count. The variation in the number of red pixels was *5–8%. The image processing was performed on a routine written in MATLAB, using a matrix that separates the RGB pixels, and counts only the red pixels of all intensities (from the lightest to the darkest). The statistical analyses for both fluorescence evaluations were performed using principal components analysis (PCA), as well as the ANOVA test (0.05%). The PCA have been used as a multidimensional statistical tool to evaluated differences between the data.6,13,14 The PCA was performed on the whole fluorescence spectrum. An algorithm was constructed for each spectrum processing method and applied for the data, using Matlab 12 platform.15 The PCA is used in most cases to investigate the dependence and connections among variables, reducing and simplifying data, and classifying them into groups.16 This technique has been applied in fluorescence spectroscopy evaluations even as in the Raman spectroscopy.17 Results
A typical fluorescence spectrum for PPIX production in the target tissue using topical application of 20% 5-ALA cream is presented in Fig. 1. The presence of PPIX is identified by two strong peaks at 633 and 700 nm.18 In
FIG. 1. Fluorescence spectrum from protoporphyrin IX (PPIX) formation kinetics in human volunteers.
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FIG. 2.
MENEZES ET AL.
Applying vacuum prototype to volunteer’s arm.
addition, use of the vacuum prototype on a volunteer is shown in Fig. 2. There are differences in the skin layers of young and old people, as the skin thickness decreases with age.19 The natural aging process of skin results as a biological change that occurs because of a decrease in the dermal–epidermal interface.13 The transdermal permeation of drugs varies according to the age of skin, skin type, the body region, and the environmental condition of the skin.20 In this study, skin aging differences related to drug permeation through the skin were not evaluated. To minimize the variability, we preferred to limit the range of volunteers’ ages. Considering the ages of our volunteers, the analyses by widefield fluorescence, using LED in 408 nm, brings us information about the amount of PPIX produced at superficial skin layers at a depth *0.1 mm or 100 lm (stratum corneum and superficial epidermis). On the other hand, the fluorescence spectroscopic analyses, using laser in 532 nm, shows us information about deeper skin layers at depth *0.4 mm or 400 lm (epidermis and epidermis–dermis junctions). In Fig. 3, we can see the PPIX formation kinetics by fluorescence spectroscopy analyses up to 7 h of treatment for
FIG. 3. Protoporphyrin IX (PPIX) formation kinetics by fluorescence spectroscopy analysis. Fluorescence intensity corresponds to the greater amplitude of PPIX band at 633 nm for each time until 7 h into the experiment. The pharmacokinetics from PPIX were obtained through increases of the band at 633 nm. Error bars are standard deviations obtained from measurement of 10 independent volunteers. all volunteers. The PPIX rate formation and overall amount formed is greater in the treatment group than in the control group. The rate formation of PPIX reaches the maximum values at 6 h of treatment, at which there were 30% differences between treatment groups. After this time, the PPIX elimination from deeper skin layers increases and overall elimination begins. Simulating the PPIX elimination curve in Fig. 3, complete elimination, in agreement with our results, occurs *12 h and 14 h in the treatment and control group, respectively (results not shown here). These results suggest that elimination from deeper tissue can occur faster in the NP-G treatment group than in the CG. In Fig. 4, we can see the PPIX formation, distribution, and elimination kinetics through qualitative evaluations, using
FIG. 4. Widefield fluorescence imaging from a volunteer during study time (24 h). Treatment group (right arm) and control group (left arm).
5-ALA, PHOTODYNAMIC THERAPY
FIG. 5. Pharmacokinetics of protoporphyrin IX (PPIX) through the skin by widefield fluorescence imaging analyses. Fluorescence intensity corresponds to the number of red pixels, from PPIX, in the image. Error bars are standard deviations obtained from measurement of 10 independent volunteers. widefield fluorescence imaging 24 h after treatment in one volunteer. The PPIX formation is faster and greater in the NP-G than in the CG. Also, the PPIX distribution is more homogeneous in the NP-G than in the CG. We believe that this occurs as a result of stimulating the net of microvessels, allowing for a more uniform production and distribution of PPIX within the considered area. As we can see in Fig. 4, the CG induces PPIX formation in lower quantities than the NP-G, and reaches a higher amount of PPIX formation in the interval between 7 and 24 h. These data suggests that the use of negative pressure decreases the photosensitivity of skin. Another aspect to emphasize is that the elimination from skin surface is faster in the NP-G than in the CG after 24 h. The complete elimination of PPIX occurs after 48 h (results not shown here). This can be beneficial because PDT can be spread over 2 days.
299
The PPIX formation kinetics by quantitative analyses using widefield fluorescence imaging 24 h after treatment for all volunteers is shown in Fig. 5. In the superficial layers, PPIX formation is faster and greater in the NP-G than in the CG, and reaches a maximum value after 6 h of treatment, and then remains constant. The results obtained by widefield fluorescence imaging in Fig. 5 showed that the differences between treatment groups is *20% at 6 h, which is comparable to our previous measurement (Fig. 3). As we can see from the results, negative pressure induction increases the blood flow and the temperature, increasing the PPIX formation levels of the skin. From infrared thermometer evaluations, the temperature increases 2 in the presence of negative pressure induction. These results are in agreement with the literature, and are connected to increasing drug permeation through the skin.18,21 In Fig. 6A it is possible to see that the autofluorescence of tissue decreases with negative pressure induction and by PCA evaluations, and in Figure 6B we prove that this change exists and is larger. Figures 7 and 8 present the statistical differences between both treatment evaluations calculated by PCA for all volunteers. Discussion
Knowing the limitations of topical PDT using ALA and its derivatives, the negative pressure induction will be useful optimizing the penetration of ALA through the skin, even as the production of PPIX at different skin layers. This happens because of an increase of blood flow rich in hemoglobin, a protein that absorbs the same range of laser wavelengths as those used to collect the fluorescence measures (see the Materials and Methods section). The negative pressure induction affects the microvascular blood flow, and can be useful in cancer treatment, optimizing the use of topical ALA in clinical PDT.10,11 In our study, we have used normal skin as the model. It is important, however, to point out that in skin cancer tissue, the overall vascular structure is very different. The relatively poor
FIG. 6. (A) Autofluorescence spectrum of protoporphyrin IX (PPIX) formation kinetics in human volunteers, using a laser in 408 and 532 nm, before and after negative pressure induction (suction). (B) Principal components analysis (PCA) using fluorescence spectroscopy analyses. The PCA of autofluorescence evaluations was performed before and after suction.
300
FIG. 7. Principal components analysis (PCA) using widefield fluorescence imaging analyses. The PCA of fluorescence evaluations was performed for both treatments [suction group (NP-G) and control group (CG)] at each time (1–7 h and 24 h) and for each volunteer (V3–V10). microvascularization of the tumor area may result in a much more accentuated effect when negative pressure is applied.22 This study suggested that the production, distribution, and elimination of PPIX are faster in the NP-G than in the CG. The results produced here demonstrated the potential clinical application of negative pressure in order to optimize topical 5-ALA-PDT. Widefield fluorescence imaging showed a greater, faster, and more homogenous increase in PPIX production at the skin surface in the NP-G than in the CG. The differences found were *20% and demonstrate that widefield fluorescence imaging is a practical technique, which can be readily applied in clinical PDT. This technique has been applied widely in our laboratory in tumor tissue diagnosis and in PDT monitoring the PPIX production, elimination, and distribution at skin.23–25
MENEZES ET AL.
The fluorescence spectroscopic technique has also been used as a diagnostic tool evaluating the spectral modifications on tissue, and can be applied in the diagnosis of tumor tissues and in liver transplantation, as well as in the evaluation of tissue modifications after death.15,26,27 In agreement with our results, the maximum amount of PPIX production, evaluated by fluorescence spectroscopic and widefield fluorescence imaging analyses, is faster and greater in the deeper layers (30%) than at the surface (20%) 6 h after the initiation of the study. Six h after the application of 20% 5-ALA cream, PPIX formation in deeper layers decreased, but remained constant in the surface layer of the NP-G, and increased for 24 h in the CG, remaining constant after that. The differences found using normal skin, 30% in spectroscopy evaluations and 20% in widefield fluorescence, were statistically significant in PCA analysis. Considering the data, we hypothesize that the differences will be *50– 60% in abnormal skin. This study has the ability to develop new procedures to improve topical PDT. After 48 h (results not shown here), the complete elimination of PPIX, from skin surface, occurs faster in the NP-G than in the CG. The fast elimination of the PS in PDT is an interesting advantage, decreasing the sensitivity of the skin, thereby minimizing this undesirable effect in PDT treatment of cancer. Certainly this procedure can be faster, amplified, when skin cancer is possible. Conclusions
In this study, we have used normal skin as the model to evaluate the benefits of negative pressure induction in topical PDT using 5-ALA. The next step in this research will be the application of negative pressure to basal-cell carcinoma (BCC) skin cancer. The differences found using normal skin, 30% in spectroscopy evaluations, and 20% in widefield fluorescence, were statistically significant in PCA. Considering these data, we hypothesize that the differences will be *50–60% in abnormal skin. This study has the ability to develop new procedures to improve the topical application of 5-ALA in PDT. In summary, our study demonstrates the high potential of negative pressure in clinical applications, optimizing PDT. Acknowledgments
The authors thank Cristina Kurachi, Fernanda Rossi Paolillo, Everton Se´rgio Estranholli, and Mardoqueu Martins da Costa, for technical assistance, and FAPESP for financial support for this research. Author Disclosure Statement
No competing financial interests exist. References
FIG. 8. Principal components analysis (PCA) using spectroscopy fluorescence analyses. The PCA of fluorescence evaluations was performed for both treatments [suction group (NP-G) and control group (CG)] at each time (1–7 h) and to each volunteer (V3–V10).
1. Agostinis, P., Berg, K., Cengel, K.A., et al. (2011). Photodynamic therapy of cancer: an update. Ca-Cancer J. Clin. 61, 250–281. 2. Henderson, B.W., Busch, T.M., and Snyder, J.W. (2006). Fluence rate as a modulator of PDT mechanisms. Lasers Surg. Med. 38, 489–493.
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3. Niedre, M.J., Secord, A.J., and Patterson, M.S. (2003). In vitro tests of the validity of singlet oxygen luminescence measurements as a dose metric in photodynamic therapy. Cancer Res. 63,7986–7994. 4. Busch, T.M., Hahn, S.M., and Evans, S.M. (2000). Depletion of tumor oxygenation during photodynamic therapy: detection by the hypoxia marker EF3[2-(2-Nitroimidazol-1[H]-yl)-N(3,3,3-trifluoropropyl)acetamide]. Cancer Res. 60, 2636–2642. 5. Uebelhoer, N.S., and Dover, J.S. (2005). Photodynamic therapy for cosmetic applications. Dermatol. Ther. 18, 242–252. 6. Lopez, R.F.V., Langeb, N., Guyc, R., and Bentley, M.V.L.B. (2004). Photodynamic therapy of skin cancer: controlled drug delivery of 5-ALA and its esters. Adv. Drug Deliv. Rev. 56, 77–94. 7. Zhang, L., Fang, Y.P., and Fang, J.Y. (2011). Enhancement techniques for improving 5-aminolevulinic acid delivery through the skin. Dermatol. Sinica. 29, 1–7. 8. Zhang, L.W., Al-Suwayeh, S.A., Hung, C.F., Chen, C.C., and Fang, J.Y. (2011). Oil components modulate the skin delivery of 5-aminolevulinic acid and its ester prodrug from oil-in-water and water-in-oil nanoemulsions. Int. J. Nanomed. 6, 693–704. 9. Dzwonczyk, R., Del Rio, C.L., and Sai-Sudhakar, C. (2006). Vacuum-assisted apical suction devices induce passive electrical changes consistent with myocardial ischemia during off-pump coronary artery bypass graft surgery. Eur. J. Cardiothorac. Surg. 30, 873–876. 10. Schintler, M.V. (2012). Negative pressure therapy: theory and practice. Diabetes Metab. Res. Rev. 28, 72–77. 11. Wackenfors, A., Sjo¨gren, J., Gustafsson, R., Algotsson, L., Ingemansson, R., and Malmsjo¨, M. (2004) Effects of vacuumassisted closure therapy on inguinal wound edge microvascular blood flow. Wound Repair Regen. 12, 600–606. 12. Vollet–Filho, J.D., Menezes, P.F.C., Moriyama, L.T., et al. (2009) Possibility for a full optical determination of PDT outcome. J. Appl. Phys. 105, 102038-1–102038-7. 13. Tosato, M.G., Alves, R.S., Dos Santos, E.A., et al. (2012) Raman spectroscopic investigation of the effects of cosmetic formulations on the constituents and properties of human skin. Photomed. Laser Surg. 30, 85–91. 14. Saraswathy, A., Jayasree, R.S., Baiju, K.V., Gupta, A.K., and Pillai, V.P. (2009) Optimum wavelength for the differentiation of brain tumor tissue using autofluorescence spectroscopy. Photomed. Laser Surg. 27, 425–433. 15. Estracanholli, E.S., kurachi, C., Vicente, J.R., Menezes, P.F.C., et al. (2009). Determination of post-mortem interval using in situ tissue optical fluorescence. Opt. Express 17, 8185–8192. 16. Venugopal, C., Nazeer, S.S., Balan, A., and Jayasree, R.S. (2013). Autofluorescence spectroscopy augmented by multivariate analysis as a potential noninvasive tool for early diagnosis of oral cavity disorders. Photomed. Laser Surg. 31, 605–612. 17. Bergholt, M.S., Zheng, W., Lin, K., Ho, K.Y., et al. (2011). Combining near-infrared-excited autofluorescence and Ra-
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man spectroscopy improves in vivo diagnosis of gastric cancer. Biosens. Bioelectron. 26, 4104–4110. Juzeniene, A., Juzenas, A., Iani, V., and Moan, J. (2007) Topical application of 5-aminolevulinic acid and its methylester, hexylester and octylester derivatives: considerations for dosimetry in mouse skin model. Photochem. Photobiol. 76, 329–334. Waller, J.M., and Maibach, H.I. (2005) Age and skin structure and function, a quantitative approach (I): blood flow, pH, thickness, and ultrasound echogenicity. Skin Res. Technol. 11, 221–235. Konda, S., Meier–Davis, S.R., Cayme, B., and Shudo, J. (2012). Age-related percutaneous penetration part 1: skin factors. Skin Therapy Lett. 17, 1–5. Yang, J., Chen, A.C.H., Wu, Q., et al. (2010). The influence of temperature on 5-aminolevulinic acid-based photodynamic reaction in keratinocytes in vitro. Photodermatol. Photoimmunol. Photomed. 26, 83–88. Pries, A.R., Cornelissen, A.J.M., Sloot, A.A., and Hinkeldey, M. (2009). Structural adaptation and heterogeneity of normal and tumor microvascular networks. PLoS Computat. Biol. 5, 1–11. Roblyer, D., Kurachi, C., Stepanek, V., Schwarz, R.A., et al. (2010) Comparison of multispectral wide-field optical imaging modalities to maximize image contrast for objective discrimination of oral neoplasia. J. Biomed. Opt. 15, 1–9. Pratavieira, S., Santos, P.L.A., Bagnato, V.S., and Kurachi, C. (2009). Development of a widefield reflectance and fluorescence imaging device for the detection of skin and oral cancer. Proc. SPIE 7380, 73805G-1–73805G-6. Inada, N., Costa, M., Ribeiro, E., Kurachi, C., Lombardi, W., and Bagnato, V.S. (2011). Protoporphyrin IX production and photobleaching during treatment of condyloma by HPV with methyl aminolevulinate. Photodiagnosis Photodyn. Ther. 8, 216–217. Castro-e-Silva, O., Sankarankutty, A.K., Correa, R.B., Ferreira, J., et al. (2008). Autofluorescence spectroscopy in liver transplantation: preliminary results from a pilot clinical study. Transplant Proc. 40, 722–725. Vollet Filho, J.D., Menezes, P.F.C., Moriyama, I.T., and Grecco, C. (2009). Possibility for a full optical determination of photodynamic therapy outcome. J. Appl. Phys. 105, 102038-1–102038-7.
Address correspondence to: Priscila Fernanda Campos Menezes Instituto de F´ısica de S~ ao Carlos (IFSC) University of S~ ao Paulo (USP) Av. Trabalhador S~ ao Carlense 400 - CEP: 13560-970 S~ ao Carlos – SP Brazil E-mail: [email protected]
Optimization of Photodynamic Therapy Using Negative Pressure Priscila Fernanda Campos Menezes, PhD, Michelle Barreto Requena, BS, and Vanderlei Salvador Bagnato, PhD
Abstract
Objective: The goal of this study is to demonstrate an alternative procedure to perform topical photodynamic therapy (PDT). Here, we propose the combined use of negative pressure and a 5-Aminolevulinic acid (5-ALA) cream occlusion to increase protoporphyrin IX (PPIX) formation. Background data: PDT using topical 5-ALA as a prodrug and precursor of PPIX has been used in the treatment and diagnosis of different types of cancer and skin diseases. The use of 5-ALA offers many advantages as a localized and non-systemic application, but it shows limitations in relation to skin penetration. Many authors have discussed the limitations of 5-ALA penetration through the skin. The skin penetration of 5-ALA can be optimized using mechanical devices associated with typical PDT procedure. Methods: For this study, 20% 5-ALA cream was applied to a 9 cm2 area of skin, and an occlusive dressing was placed. The PPIX production was collected at the skin surface, using fluorescence spectroscopy and widefield fluorescence imaging, for 7 h, and after 24 h. Results: We observed that in the presence of negative pressure therapy, the PPIX production, distribution, and elimination are greater and faster than in the control group. The PPIX formation was *30% in deeper skin layers, quantified by fluorescence spectroscopy analysis, and *20% in surface skin layers, quantified by widefield fluorescence imaging analysis. Conclusions: Negative pressure induction can also help PDT application in the case of inefficient PPIX production. These results can be useful for optimizing the PDT. Introduction
P
hotodynamic therapy (PDT) is a procedure that exploits the consequences originating from localized oxidative damage inflicted by photochemical processes.1 There are three critical elements required for PDT: a drug that can be activated by light [a photosensitizer (PS)], light, and oxygen. Light interacting with a PS produces an excited triplet state, which interacts with ground state oxygen creating singlet oxygen (102).1–3 The highly reactive singlet oxygen promotes oxidative damage in the immediate vicinity of the subcellular site of a localized PS.3 The tissue response to PDT depends upon adequate tumor oxygenation, as well as sufficient accumulation of a PS.4 PDT, using 5-Aminolevulinic acid (5-ALA), as a precursor of protoporphyrin IX (PPIX), has been used in the treatment and diagnosis of different types of cancer, skin diseases, and skin aging.1,5 5-ALA is found naturally in our body, and is converted into PPIX, the immediate precursor of heme. Because of the feedback mechanism, the excess exogenous 5-ALA results in intracellular accumulation of PPIX. PPIX is the endogenous PS used in topical PDT.6
5-ALA can permeate easily into the epidermis of cancerous tissue, because of altered metabolism, but not normal epidermis, thus creating highly selective photosensitivity in tumor tissue.1 All topical application interference involving 5-ALA during PDT can be summarized accordingly: problems related to chemical and physicochemical characteristics of 5-ALA, problems with the light system (ideal fluence), tumor surface characteristics (tumor depth and skin thickness), adjuvant mechanical techniques used in the treatment, and increasing the temperature of the skin.7 Therefore, 5-ALA penetration through skin depends upon the optimization of all these parameters.6–8 Another important aspect, normally not considered, is the condition of tissue in relation to its ability to convert 5- ALA in PPIX. Negative pressure therapy, also known as vacuum therapy, has been used to heal wounds.9,10 The negative pressure affects the microvascular blood flow by increasing the blood flow. This alteration in the blood flow occurs because of modification in the vascular diameter, blood flow velocity, blood volume, and others things.10,11 The purpose of this work is to show that it is possible to increase the formation, distribution, and elimination of PPIX, produced by its precursor 5-ALA through the skin,
Instituto de Fı´sica de Sa˜o Carlos (IFSC), University of Sa˜o Paulo (USP), Sa˜o Carlos - SP, Brazil.
296
5-ALA, PHOTODYNAMIC THERAPY
using negative pressure. The possibility of increasing the amount of PPIX production, decreasing the interval between the application of 5-ALA cream, and irradiation time, can be useful to clinical PDT in transdermal applications. In conclusion, the negative pressure can also help PDT application in the case of inefficient PPIX production, as it grows the production and uniformity of PPIX in the skin, increasing the effectiveness of PDT. Materials and Methods Chemicals
The studies were done using 5-Aminolevulinic acid hydrochloride (5-ALA) with a 20% concentration, in an oilin-water emulsion (O/W) obtained from PDT-PHARMA (Brazil). The formulation was evenly applied to a circular area of 9 cm2 with a sterile spatula, using a density of 32 mg/cm2. The area was dressed with an occlusive mask to protect it from light. Equipment for negative pressure induction
The vacuum prototype used to stimulate PPIX production in the skin can be adjusted from 0 to 550 mm Hg (negative pressure), operating in continuous or pulsed mode. This study was performed using 200 mm Hg for 4 min, during which we alternated between continuous mode (5 sec) and pulsed mode (55 sec). The temperature increases in the skin were evaluated using an infrared thermometer (ST-600, Incoterm, Sa˜o Paulo) at 1 h intervals. Volunteers: inclusion and exclusion criteria
The protocol used in this study followed the procedures establish by Human Research Ethics Committee of Brazil. Written informed consent was obtained from all participants. Ten women patients, *25 years of age with a clinical diagnosis of normal skin, were recruited for this clinical study. All patients had Fitzpatrick skin types II (50%) or III (50%). To be considered eligible, a patient had to be free of skin disorders on both arms. Patients with lesions in the target area or with porphyria were excluded. Additional exclusion criteria included pregnancy, lactation, an allergy to 5-ALA, male gender, and being < 20 or > 35 years of age. Treatment procedure
The study was randomized with two treatment groups. The first treatment group was treated with 20% 5-ALA cream and a vacuum device for negative pressure induction (NP-G). For the second treatment group, the control group (CG), treatment was performed on the volunteers’ other arm. Autofluorescence of the skin was used as the control for each group. Comparisons were made for each treatment group and between both treatments. First, the 5-ALA cream and the vacuum device were applied for 4 min. Following this, the fluorescence was measured at the skin surface using a spectrometer, and pictures of the area were taken using widefield fluorescence imaging equipment, as previously described. At the end of the fluorescence analysis, 5-ALA cream was reapplied to the treatment area and covered with an occlusive dressing. The same procedure was performed on the other arm without the use of the vacuum device.
297 Fluorescence spectroscopy
Fluorescence collection was done using a system composed of a doubled Nd:YAG laser emitting at 532 nm, and a spectrometer, collecting fluorescence in the range of 400– 1000 nm as previously described by us.12 For fluorescence spectrum analysis, we collected the fluorescence spectrum in contact with the tissue at five points in the 9 cm2 circular area. At each point, 20 fluorescence and autofluorescence measurements were performed. The evaluations from spectra analyses were performed using the maximum value of the fluorescence spectrum (at 633 nm) from PPIX for 7 h. The spectrum evaluations were performed using Matlab 12 platform (The MathWorks, USA). Widefield fluorescence imaging
For this study, commercial equipment called ‘‘Evince’’ was used (MM Optics, Brazil), which is composed of a lighting device based on LEDs, emitting *400 and 450 nm, a high-pass filter, dichroic mirrors, a band pass filter, and a CCD camera for image acquisition. The widefield fluorescence imaging was collected at the skin surface at each hour, from 1 to 7 h, and in 24 h. The analysis was performed by collecting five images of the treatment area each hour and evaluating the pixel count. The variation in the number of red pixels was *5–8%. The image processing was performed on a routine written in MATLAB, using a matrix that separates the RGB pixels, and counts only the red pixels of all intensities (from the lightest to the darkest). The statistical analyses for both fluorescence evaluations were performed using principal components analysis (PCA), as well as the ANOVA test (0.05%). The PCA have been used as a multidimensional statistical tool to evaluated differences between the data.6,13,14 The PCA was performed on the whole fluorescence spectrum. An algorithm was constructed for each spectrum processing method and applied for the data, using Matlab 12 platform.15 The PCA is used in most cases to investigate the dependence and connections among variables, reducing and simplifying data, and classifying them into groups.16 This technique has been applied in fluorescence spectroscopy evaluations even as in the Raman spectroscopy.17 Results
A typical fluorescence spectrum for PPIX production in the target tissue using topical application of 20% 5-ALA cream is presented in Fig. 1. The presence of PPIX is identified by two strong peaks at 633 and 700 nm.18 In
FIG. 1. Fluorescence spectrum from protoporphyrin IX (PPIX) formation kinetics in human volunteers.
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FIG. 2.
MENEZES ET AL.
Applying vacuum prototype to volunteer’s arm.
addition, use of the vacuum prototype on a volunteer is shown in Fig. 2. There are differences in the skin layers of young and old people, as the skin thickness decreases with age.19 The natural aging process of skin results as a biological change that occurs because of a decrease in the dermal–epidermal interface.13 The transdermal permeation of drugs varies according to the age of skin, skin type, the body region, and the environmental condition of the skin.20 In this study, skin aging differences related to drug permeation through the skin were not evaluated. To minimize the variability, we preferred to limit the range of volunteers’ ages. Considering the ages of our volunteers, the analyses by widefield fluorescence, using LED in 408 nm, brings us information about the amount of PPIX produced at superficial skin layers at a depth *0.1 mm or 100 lm (stratum corneum and superficial epidermis). On the other hand, the fluorescence spectroscopic analyses, using laser in 532 nm, shows us information about deeper skin layers at depth *0.4 mm or 400 lm (epidermis and epidermis–dermis junctions). In Fig. 3, we can see the PPIX formation kinetics by fluorescence spectroscopy analyses up to 7 h of treatment for
FIG. 3. Protoporphyrin IX (PPIX) formation kinetics by fluorescence spectroscopy analysis. Fluorescence intensity corresponds to the greater amplitude of PPIX band at 633 nm for each time until 7 h into the experiment. The pharmacokinetics from PPIX were obtained through increases of the band at 633 nm. Error bars are standard deviations obtained from measurement of 10 independent volunteers. all volunteers. The PPIX rate formation and overall amount formed is greater in the treatment group than in the control group. The rate formation of PPIX reaches the maximum values at 6 h of treatment, at which there were 30% differences between treatment groups. After this time, the PPIX elimination from deeper skin layers increases and overall elimination begins. Simulating the PPIX elimination curve in Fig. 3, complete elimination, in agreement with our results, occurs *12 h and 14 h in the treatment and control group, respectively (results not shown here). These results suggest that elimination from deeper tissue can occur faster in the NP-G treatment group than in the CG. In Fig. 4, we can see the PPIX formation, distribution, and elimination kinetics through qualitative evaluations, using
FIG. 4. Widefield fluorescence imaging from a volunteer during study time (24 h). Treatment group (right arm) and control group (left arm).
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FIG. 5. Pharmacokinetics of protoporphyrin IX (PPIX) through the skin by widefield fluorescence imaging analyses. Fluorescence intensity corresponds to the number of red pixels, from PPIX, in the image. Error bars are standard deviations obtained from measurement of 10 independent volunteers. widefield fluorescence imaging 24 h after treatment in one volunteer. The PPIX formation is faster and greater in the NP-G than in the CG. Also, the PPIX distribution is more homogeneous in the NP-G than in the CG. We believe that this occurs as a result of stimulating the net of microvessels, allowing for a more uniform production and distribution of PPIX within the considered area. As we can see in Fig. 4, the CG induces PPIX formation in lower quantities than the NP-G, and reaches a higher amount of PPIX formation in the interval between 7 and 24 h. These data suggests that the use of negative pressure decreases the photosensitivity of skin. Another aspect to emphasize is that the elimination from skin surface is faster in the NP-G than in the CG after 24 h. The complete elimination of PPIX occurs after 48 h (results not shown here). This can be beneficial because PDT can be spread over 2 days.
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The PPIX formation kinetics by quantitative analyses using widefield fluorescence imaging 24 h after treatment for all volunteers is shown in Fig. 5. In the superficial layers, PPIX formation is faster and greater in the NP-G than in the CG, and reaches a maximum value after 6 h of treatment, and then remains constant. The results obtained by widefield fluorescence imaging in Fig. 5 showed that the differences between treatment groups is *20% at 6 h, which is comparable to our previous measurement (Fig. 3). As we can see from the results, negative pressure induction increases the blood flow and the temperature, increasing the PPIX formation levels of the skin. From infrared thermometer evaluations, the temperature increases 2 in the presence of negative pressure induction. These results are in agreement with the literature, and are connected to increasing drug permeation through the skin.18,21 In Fig. 6A it is possible to see that the autofluorescence of tissue decreases with negative pressure induction and by PCA evaluations, and in Figure 6B we prove that this change exists and is larger. Figures 7 and 8 present the statistical differences between both treatment evaluations calculated by PCA for all volunteers. Discussion
Knowing the limitations of topical PDT using ALA and its derivatives, the negative pressure induction will be useful optimizing the penetration of ALA through the skin, even as the production of PPIX at different skin layers. This happens because of an increase of blood flow rich in hemoglobin, a protein that absorbs the same range of laser wavelengths as those used to collect the fluorescence measures (see the Materials and Methods section). The negative pressure induction affects the microvascular blood flow, and can be useful in cancer treatment, optimizing the use of topical ALA in clinical PDT.10,11 In our study, we have used normal skin as the model. It is important, however, to point out that in skin cancer tissue, the overall vascular structure is very different. The relatively poor
FIG. 6. (A) Autofluorescence spectrum of protoporphyrin IX (PPIX) formation kinetics in human volunteers, using a laser in 408 and 532 nm, before and after negative pressure induction (suction). (B) Principal components analysis (PCA) using fluorescence spectroscopy analyses. The PCA of autofluorescence evaluations was performed before and after suction.
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FIG. 7. Principal components analysis (PCA) using widefield fluorescence imaging analyses. The PCA of fluorescence evaluations was performed for both treatments [suction group (NP-G) and control group (CG)] at each time (1–7 h and 24 h) and for each volunteer (V3–V10). microvascularization of the tumor area may result in a much more accentuated effect when negative pressure is applied.22 This study suggested that the production, distribution, and elimination of PPIX are faster in the NP-G than in the CG. The results produced here demonstrated the potential clinical application of negative pressure in order to optimize topical 5-ALA-PDT. Widefield fluorescence imaging showed a greater, faster, and more homogenous increase in PPIX production at the skin surface in the NP-G than in the CG. The differences found were *20% and demonstrate that widefield fluorescence imaging is a practical technique, which can be readily applied in clinical PDT. This technique has been applied widely in our laboratory in tumor tissue diagnosis and in PDT monitoring the PPIX production, elimination, and distribution at skin.23–25
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The fluorescence spectroscopic technique has also been used as a diagnostic tool evaluating the spectral modifications on tissue, and can be applied in the diagnosis of tumor tissues and in liver transplantation, as well as in the evaluation of tissue modifications after death.15,26,27 In agreement with our results, the maximum amount of PPIX production, evaluated by fluorescence spectroscopic and widefield fluorescence imaging analyses, is faster and greater in the deeper layers (30%) than at the surface (20%) 6 h after the initiation of the study. Six h after the application of 20% 5-ALA cream, PPIX formation in deeper layers decreased, but remained constant in the surface layer of the NP-G, and increased for 24 h in the CG, remaining constant after that. The differences found using normal skin, 30% in spectroscopy evaluations and 20% in widefield fluorescence, were statistically significant in PCA analysis. Considering the data, we hypothesize that the differences will be *50– 60% in abnormal skin. This study has the ability to develop new procedures to improve topical PDT. After 48 h (results not shown here), the complete elimination of PPIX, from skin surface, occurs faster in the NP-G than in the CG. The fast elimination of the PS in PDT is an interesting advantage, decreasing the sensitivity of the skin, thereby minimizing this undesirable effect in PDT treatment of cancer. Certainly this procedure can be faster, amplified, when skin cancer is possible. Conclusions
In this study, we have used normal skin as the model to evaluate the benefits of negative pressure induction in topical PDT using 5-ALA. The next step in this research will be the application of negative pressure to basal-cell carcinoma (BCC) skin cancer. The differences found using normal skin, 30% in spectroscopy evaluations, and 20% in widefield fluorescence, were statistically significant in PCA. Considering these data, we hypothesize that the differences will be *50–60% in abnormal skin. This study has the ability to develop new procedures to improve the topical application of 5-ALA in PDT. In summary, our study demonstrates the high potential of negative pressure in clinical applications, optimizing PDT. Acknowledgments
The authors thank Cristina Kurachi, Fernanda Rossi Paolillo, Everton Se´rgio Estranholli, and Mardoqueu Martins da Costa, for technical assistance, and FAPESP for financial support for this research. Author Disclosure Statement
No competing financial interests exist. References
FIG. 8. Principal components analysis (PCA) using spectroscopy fluorescence analyses. The PCA of fluorescence evaluations was performed for both treatments [suction group (NP-G) and control group (CG)] at each time (1–7 h) and to each volunteer (V3–V10).
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Address correspondence to: Priscila Fernanda Campos Menezes Instituto de F´ısica de S~ ao Carlos (IFSC) University of S~ ao Paulo (USP) Av. Trabalhador S~ ao Carlense 400 - CEP: 13560-970 S~ ao Carlos – SP Brazil E-mail: [email protected]
