1. Introduction Improvement in both treatment and diagnosis of cancer is a constant motivation for the scientific community. Photodynamic Therapy (PDT) is a local treatment modality based on three main components, namely light, a photosensitive compound and oxygen present in both the cell and the microenvironment. The interaction between the light and the photosensitizer (PS) results in the excited state of PS that will react mainly with the oxygen. The singlet oxygen produced is highly reactive and toxic to the cell. (1–5) Dosimetry studies are very important for the therapy effectiveness since minimum doses of the three elements of PDT are necessary and involve, basically, three factors, namely light distribution in the tissue, variations on PS concentration and tissue oxygenation. Oxygen depletion during the PDT irradiation is directly related to the induced damage on the 1

vascularization network and plays an important role in the overall final photodynamic response. The dynamic of PS distribution and the time necessary for a sufficient concentration in the target tissue are also parameters to be considered. (6,7) Fluorescence is a light emission process that results from the energy release of the lower vibrational level of an excited state of a molecule after photon absorption or intermolecular interactions, when the electronic state transition is permitted. (8,9) Most diagnosis techniques that use fluorescence are non-invasive and are considered attractive tools for the detection of diseases and fluorescent compounds. (10) Protoporphyrin IX (PpIX) and Photogem® are types of PS that accumulate, preferentially, in tumor cells and have specific fluorescence emission. Therefore, these PS can be used as markers for cancer detection and visualized by fluorescence imaging, in a type of detection called Photodynamic Detection. (11,12) The individual understanding of PDT and fluorescence effects in each part of the organism is important for improvements in the techniques. Animal models show the global response of the organism to the treatment of diseases as cancer. However, they enable no individual analysis of therapies mechanisms isolated from other factors since several side effects induced by the therapy or the conditions created to mimic it or even defense mechanisms can disrupt the analysis of the response in individualized structures. Chorioallantoic membrane (CAM) is a vascular model used in pre-clinical studies on PDT that involve tests with photosensitizers and distinct PS deliveries. (13–16) The introduction of a tumor in this environment enables the evaluation of PDT mechanisms and it is used as a model to understand the effects on the blood vessels (17), characterization of tumor response more complex than cell culture (18) and vascular regrowth during and after PDT. (19) There are three distinct ways for the action of PDT: a direct cellular effect, the vascular effect and the effect of immune system. Therefore, a model that enables the monitoring of mechanisms of cell death, angiogenesis and destruction of blood vessels that feed the tumor is essential for the understanding of the PDT effects and improvements in the therapeutic efficiency. The authors aimed to reach a deeper understanding on PDT effects by the fluorescence assessment with a tumor model in CAM. From this, the comprehension about the dynamic distribution of ALA and Photogem® using a simple model with a simple image processing can enable the evaluation of PDT response for several parameters and could be the first step to analyze different and new photosensitizers.

2. Material and Methods

2.1 Ehrlich Tumor in the CAM model The CAM model in chicken eggs is an established model that enables a direct access to blood vessels. Eggs were obtained at the first day of embryo development (ED) and were maintained at 37.7°C in a humid environment. On ED 3, a window of approximately 2 cm2 was opened in the shell by tweezers and an adhesive tape sealed the hole. The tumor development is started with the introduction of a small piece of biopsied tumor, on ED 4 to have no stress of the embryo in the same day. All procedures were conducted inside the laminar flow chamber to avoid egg contamination. The solid tumor was obtained by Ehrlich model in Hairless mouse (20), after a subcutaneous injection of 0.5 mL of ascitic form. Approximately ten days after inoculation, the animals were euthanized with CO2 and the solid tumor was removed and immersed in a solution with antibiotic and saline solution. Each tumor was cut into small pieces (of approximately 100 mg and maximum thickness of 5 mm) with the aid of a scalpel, avoiding to select non-viable parts of the tumor (without vascularization). Normally, the aspect of tumor indicates if it is viable or not, since when there is necrosis, the tumor disintegrates easily and there is no indication of vascularization. These samples were discarded and only pieces with firm consistency and

2

blood vessel formation were used. A tissue slice was placed on the CAM model on the 4th day. (21) Each egg was treated with 100 µL of a solution with 10% penicillin and streptomycin antibiotics each 2 days. Experiments were performed on the ED14, after 10 days of placement of the tumor at the CAM and there is a viable interaction after this time. However, after two days it was already possible to observe the beginning of the vessels in the tumor environment. All procedures were approved by the Institutional Animal Care and Use Committee of Sao Carlos Institute of Physics (protocol number 9/2014) and Institutional Animal Care and Use Committee of Federal University of Sao Carlos (protocol number 068/2012). The eggs were frozen at -20°C, before being discarded.

2.2 Photosensitizers and PDT Parameters Two PS were investigated - Photogem® (Photogem, Russia), which is a porphyrin-based commercial photosensitizer used in the clinical application of PDT and PpIX, which is an endogenous porphyrin-based PS produced by the application of pro-drug Aminolevulinic Acid (ALA, PDTPharma, Brazil). Both compounds were diluted in distilled water for the obtaining of the desired concentration using a volume of 200 µL, i.e., 10% for ALA and 10 mg/L for Photogem®. The incubation time when PDT was applied was 4 h so that the results from both could be compared. Solutions were topically and intravenously administered, initially. Three different groups were performed for each PS and three eggs per group were used for the analysis of different effects. Firstly, only PS was applied on the CAM for the observation of the interaction between the molecule and the vessels. PS was then placed in the tumor model for the analysis of the dynamics of PS and the tumor without light. Finally, after the understanding of these groups, PDT was applied to the tumor model. Considering previous studies only in blood vessels, (17) the area of irradiation was delimited using a Teflon® ring by 1.76 cm2 for PDT group and the spot of laser was the same of this area to result in 80 mW/cm2, for 10 minutes which totaled 48 J/cm2 dose light with a diode laser (Quantum Tech, São Carlos, Brazil) centered at 635 nm. Two control groups (only light and only saline solution) were performed to ensure all effects observed resulted from PS or PDT.

2.3 Image acquisition Fluorescence images were acquired by a CCD camera (PIXELFLY QE – PCO, Germany) and a fluorescence visor (VELSCOPE, USA) that emits light in wavelengths between 400 and 460 nm and has optical filters for highlighting the visualization of the red emission, ideal for the PpIX and Photogem® detection. The camera and fluorescence device were positioned over the eggs, in front of the window in the shell for the direct visualization of the tumor in the CAM. Figure 1 shows how the setup was done. All components, except the computer that was connected to the camera, were kept inside the laminar flow chamber. CamWare® (CamWare, PCO, Germany) software was used for the image acquisition. Images were acquired from zero to 5 h, 8 h, 24 h, 48 h or 75 h, depending on the kinetics of the PS production/distribution. However, the eggs were removed from the incubator only for the image acquisition and were repositioned in the incubator for the avoidance of damage due to the temperature changes.

2.4 Analysis of the images The temporal monitoring of fluorescence was performed by acquiring the widefield fluorescence images of the CAM. The biological tissues show a native fluorescence, called autofluorescence, with the main emission in the green region of the spectrum, especially due 3

to aminoacids and NADH and FAD, electron carriers of the respiratory chain. On the other hand, porphyrins emit a red fluorescence that can be discriminated within the tissue or the vessel. The aim of the present study was to monitor the PS distribution and photobleaching considering the tumor vessels and cells. The most relevant image data, therefore, is the pixel intensity of the red channel as it is used in the literature to quantify PpIX. (22,23) The exposure time of each image was ranged between 25 ms and 65 ms and recorded for intensity normalization in the algorithm of image analysis. This procedure was necessary because the red signal is low in the autofluorescence images and a higher exposure time was required. However, with an increased porphyrin emission, this value was changed for the avoidance of saturation. The red pixel counting was evaluated by an algorithm developed in MATLAB® software (MATrix LABoratory, MathWorks, USA). First, a region of interest (ROI) was extracted from each image by Photoshop® (ADOBE PHOTOSHOP CS4, version 11, USA) and GIMP (GNU Image Manipulation Program) software. The images were uploaded and only the red channel data was used. As both studied photosensitizers show a red fluorescence and major changes were observed in this channel when compared to control study without PS, so only this information was chosen to quantify the formation or bleaching of our compounds. The images were scanned and the sum of the values of all the red pixels and the number of pixels in the ROI were computed. Different investigation times were compared for the evaluation of the mean value of the red pixels, set by the ratio between the number of red pixels and total number of the pixels in each image. The calculated values were plotted according to each image to show the correlation of the percentage of red pixels and interrogated time. OriginPro® 8.1 SR3 (OriginLab Corporation, EUA) was used for the data analysis of data and graphics. The value of the coefficient of determination (R-square) was used for each graph for the adjustment of data fitting.

3. Results and Discussion The monitoring of the pixels associated to red fluorescence remained constant over time for control groups (data not shown), confirming the constant behavior and ensuring that the red pixel counting is related to PS actions.

3.1 PpIX production - ALA On the initial tests, both ways of PS administration showed similar results considering the PS dynamic distribution and incubation time. However, as the intravenous injection can cause some hemorrhage and damage to the vascular network, the topical administration was chosen for further experiments.

3.1.1 Fluorescence in the Blood Vessels Initially, the dynamics of the photosensitizers distribution was analyzed only in the vasculature network. The control group (without light and PS) showed no alteration in the fluorescence images. Figure 2 shows a sequence of images with the topical application of ALA. The first image (0 h) shows the CAM autofluorescence where the membrane and albumin show a green emission and the vessels are discriminated as darker reddish long structures. After 3 h of ALA application, the larger vessels on the surface showed an increased red fluorescence, more intense in the endothelial cells, which indicated the production of PpIX. The PpIX concentration increased after a longer time and was detected by the brighter red fluorescence of the vessels and even within the CAM, as observed after 75 h. ALA diffuses within the CAM, the cells and vessels at the superficial layers are the first ones to absorb the pro-drug molecule and start the PpIX production. Endothelial cells, which compose the blood vessel walls, naturally have the biosynthesis of PpIX molecule in the mitochondria and were the first site to show its production and accumulation. PpIX 4

production becomes systemic, even by the embryo, due both the diffusion of ALA through all CAM and its whole egg distribution through the vessels. Figure 3 shows the red pixel counting by fluorescence emission of the CAM as a function of time for the topical ALA application. The increase in the PpIX concentration showed an exponential behavior in all treated eggs. As the control groups (without ALA) did not show this behavior, it is possible to suggest that this increase is because of PpIX production. The following equation was used for the analysis of the PpIX dynamics (1): t −𝑡 𝜏

𝐼(𝑡) = 𝐼0 (1 − 𝑒 ) + 𝑘

I (t )  I 0 (1  e )  k 

(1)

where I and t correspond to the intensity of red fluorescence (arbitrary units) and time (h), respectively, and I0 is the intensity of red native fluorescence at t=0, before ALA application. (32) τ is the characteristic time of the PpIX formation and a parameter that enables the comparison of the results, which higher value means longer time for the detection of the saturation level of PpIX. Parameter k was added to the equation in order to consider in different samples, inherent differences of I0. Origin® fitted the experimental data obtained by the measurement of the fluorescence intensity over time and the corresponding curve of equation (1) with values for the parameter τ, I0 and k, characterizing the dynamic behavior of the PpIX formation. Using the example of the red fluorescence behavior presented in Figure 3, it is possible to observe, during the first 7 h, the PpIX production occurs with an almost linear temporal relation and the saturation starts after around 10 h. After 75 h of ALA incubation, the mean value of τ was (8.3 ± 3.6) h. The PpIX production shows a higher rate in the first 6 h, observed mainly within the vessels. At longer investigated times, fluorescence was observed in all CAM, resulting from an overall sensibilization of the embryo and egg structures. No decrease in the PpIX fluorescence emission was observed up to 75 h. The mean value of R-Square was (91 ± 5) %, showing that the fitting curve was strongly correlated with the experimental data.

1.1.1 Fluorescence within the tumor The PS distribution in the tumor cells and vessels must be analyzed for a better evaluation of the PDT response in a tumor environment. In an ALA topical application, the kinetics of PpIX production can be distinct, when considering the tumor or endothelial cells; therefore, the tumor in the CAM model was investigated. Figure 4 shows the qualitative analysis of the PpIX production as a function of time after the topical application of ALA. The red fluorescence in the tumor region in 1.5 h is more pronounced in the vessels and increased for longer times. PpIX is produced firstly in the vessels because these are the first structures that ALA molecules interact with, and the

5

delayed PS concentration in the tumor may be a result of a later tumor cell production or the systemic PpIX distribution through the vessels. After 24 h, even with PpIX distribution in all CAM structures, the highest concentration could be observed within the tumor region. The same analysis of the PpIX production after ALA application for the whole CAM and for the tumor region of the CAM alone was performed, and then, the results were compared. The production in the whole CAM, as showed in the graph, resulted in a mean τ of (6.7 ± 2.5) h. To evaluate the difference between the PpIX production within the tumor and the whole CAM, the variance in τ (Δτ) was calculated, as showed in equation 2. (2) Δτ = τ tumor region – τ CAM+tumor The resulted Δτ= (2.1 ± 1.6) h may indicate an increase of τ and, therefore, that the production time is longer within this region. Such values corroborated with the clinical characteristics that ALA is more concentrated in the tumor cells and the vessels produced in the first hours at a higher rate. As expected, the value of τ in the analysis of membrane with the tumor is lower than CAM without tumor, since the PpIX production is concentrated only in one region. The mean R-square was (88 ±7) %, which can be considered a high value, especially for in vivo investigation between fitted and experimental data. Clinical studies (24) and animal tests (25) showed a similar behavior of PpIX production, with the fluorescence peak of production between 4 and 6 h of application. These results support the validity of the used methods and analysis.

1.2 Photogem®

1.2.1 Fluorescence in the Blood Vessels The topical application of Photogem® was also chosen as the administration via to avoid any vessel damage and further changes in the vasculature structure. Figure 5 shows the temporal distribution of the PS. Immediately after the topical application of the porphyrin solution (0 h), a characteristic red fluorescence was observed; however, in this case, the photosensitizer was not inside the cells or vessels. After 30 minutes, due to the diffusion through the membrane, all CAM structures showed red fluorescence. For longer incubation time, the fluorescence was observed in both vessels and the embryo. The graph of Figure 6 clearly shows this inverse behavior in comparison to ALA. This was expected, since, differently from the ALA-formed PpIX, Photogem® is a photosensitizer delivered in its final form, and requires no time to be produced in the cells. Only the diffusion of the molecule through the CAM and its interaction with the epithelial cells of blood vessels are necessary. The molecules of both PS have different sizes; therefore, one of the consequences is that the interaction between the compound and the cell membrane is different. Part of porphyrin is degraded and the rest is transported by the blood flow, which increases the diffusion for all CAM and embryo and gradually decreases its concentration in the blood vessels. The best adjustment curve for the data is an exponential decay function: 6

t

𝐼(𝑡) = 𝐼0 𝑒

−𝑡 𝜏

+𝑘

(3)

I (t )  I 0 e  k 

where I also corresponds to the fluorescence value, t denotes the time in h, I0 is the value of initial fluorescence intensity and k is a constant for the best adjustment. In this case, the mean value of τ was (2.8 ± 1.4) h and in the first hours, the highest fluorescence was observed. However, Photogem® is not inside the vessels and the light application in this time interval could not result in blood vessels destruction. With ALA application, there is not this problem because the production is inside the endothelial cells, and so, the PS. The mean R-square value was (87±4) %, showing that the fitting equation could explain the experimental Photogem® data.

1.2.2 Fluorescence within the tumor The comparison between the PS molecules shows Photogem® is composed of a mixture of monomers, dimmers and oligomers, and has greater effectiveness when applied by intravenous way because this compound has big molecules and, consequently, presents greater difficulty in crossing cell barriers. On the other hand, ALA is a small molecule and presents a better diffusion through the CAM in comparison to porphyrins in different molecular forms, which results in a more homogenous PpIX distribution. As it is a prodrug, ALA has no photodynamic activity and is more used in topic application, facilitating its application in this model. Although a higher PS concentration was observed in the tumor region for Photogem®, its distribution was more heterogeneous. The same type of image analysis was performed using equation 3. The mean τ determined for the whole region was (4.5 ± 0.9) h; however, the mean Δτ for the tumor region was of (4.1 ± 1.4) h, higher than τ for all CAM, which shows that the decay occurs slowly within the tumor region. Photogem® was accumulated in the tumor and the value of τ is different in the region with and without tumor, as observed for ALA. However, since the behavior of both PS is opposite, the value of τ for Photogem® without tumor is lower than the tumor model. The mean R-Square was (79±9) % - all values are summarized in table 1. The parameters for the application of PDT were defined according to such values.

1.3 Photodynamic Therapy in the Tumor Model Using topical application of ALA, Figure 7 shows the monitoring of PpIX fluorescence over the time. After a 4h incubation, PpIX was concentrated around the tumor and, immediately after PDT illumination, a high decrease in the red fluorescence indicated the superficial PS photobleaching. This incubation time was chosen because it is within the period in which the PS production in the tumor has not stabilized. After 7.5 h of PDT treatment, which corresponds to 11.5 h of the experiment, a PS concentration was visualized in the embryo. After PDT, partial vasculature damage was observed. The graph of Figure 8 shows the fluorescence quantification with a clear decrease after illumination. As ALA remains in the environment, the viable cells still produce PpIX and, consequently, an increase in the red fluorescence could be observed after 3-4 h. The graph shows three distinct regions, namely I -before PDT (0-4 h), II- Immediately after PDT (4-7 h) and III- Post-PDT – secondary PpIX production (7-48 h). 7

Graphs with adjustments in each part were plotted and the equation 1 was used to monitor the production of PpIX for regions I and III, with calculated τ of 1.84 h and 17.5 h, respectively. The highest value for the last region is due the production of PS started in the embryo which is not of our interest for PDT application. The reappearance of fluorescence between 1 and 3 h after the light application is observed in several other studies confirming the presence of ALA which has not been metabolized and is still present in the tissues. (8,26) The tumors acquired a different aspect in comparison to their initial appearance with loss of tissue integrity and no connections with the blood vessels. Such characteristics indicate effective PDT response. Photogem® was used for PDT analysis following the same protocol of ALA and, therefore, the same incubation time, topical application and total light dose. As expected, initially, the fluorescence was more concentrated in the tumor and surroundings and, after light application; almost all PS had been consumed. Differently from ALA, after PDT, there is no secondary fluorescence increase because Photogem® was directly delivered and it is not produced. Both ALA-PDT and PhotogemPDT protocols showed a loss of tumor tissue integrity indicating a tumor destruction.

4. Conclusions CAM model could be a great option to study the interaction between therapies parameters and the tumor with vessel. The dynamic behavior of ALA-PpIX in the blood vessels of a CAM/tumor model was determined through an equation that describes the PpIX production accurately (R-Square around 90%). The tumor developed in the CAM enables a simple model to understand the interaction of different PS with blood vessels and tumor cells. Photogem® showed an opposite behavior in comparison to the production of PpIX since, after its application, it is internalized in tumor and endothelial cells and is photobleached after PDT irradiation. A higher PS accumulation was observed within the tumor region and an efficient photodynamic response was achieved with both photosensitizers. Acknowledgements The authors acknowledge the financial support provided by FAPESP (CEPOF-CEPID Program) and CAPES to HH Buzzá scholarship and A’doro SA enterprise for supplying all eggs. References [1] Wilson BC,;1; Patterson MS. The physics, biophysics and technology of photodynamic therapy. Phys Med Biol. 2008;53(9):R61–109. [2] Ackroyd R, Kelty C, Brown N, Reed M.;1; The history of photodetection and photodynamic therapy. Photochem Photobiol. 2001;74(5):656–69. [3] Rechtman E, Ciulla T a, Criswell MH, Pollack A, Harris A.;1; An update on photodynamic therapy in age-related macular degeneration. Expert Opin Pharmacother. 8

2002;3(7):931–8. [4] Triesscheijn M, Baas P, Schellens JHM, Stewart F;1;a. Photodynamic therapy in oncology. Oncologist. 2006;11(9):1034–44. [5] Dougherty T., Gomer C., Henderson B., Jori G., Kessel D., Korbelik M., Moan J.;1; PQPT. Photodynamic therapy. J Natl Cancer Inst. 1998;12(90)(12):889–905. [6] Zhou X, Chen B, Hoopes PJ, Hasan T, Pogue BW.;1; Tumor vascular area correlates with photosensitizer uptake: analysis of verteporfin microvascular delivery in the Dunning rat prostate tumor. Photochem Photobiol. 2006;82(5):1348–57. [7] Vollet-Filho JD, Caracanhas MA, Grecco C, Ferreira J, Kurachi C, Bagnato VS.;1; Nonhomogeneous liver distribution of photosensitizer and its consequence for photodynamic therapy outcome. Photodiagnosis Photodyn Ther. 2010;7(3):189–200. [8] Juzenas P, Sharfaei S, Moan J, Bissonnette R.;1; Protoporphyrin IX fluorescence kinetics in UV-induced tumours and normal skin of hairless mice after topical application of 5aminolevulinic acid methyl ester. J Photochem Photobiol B Biol. 2002;67(1):11–7. [9] Svanberg K, Klinteberg C, Nilsson A, Wang I, Andersson- Engels S, Svanberg S.;1; Laser-based spectroscopic methods in tissue characterization. Ann NY Acad. 1998;838:123–9. [10] Pottier RH, Chow YF, LaPlante JP, Truscott TG, Kennedy JC, Beiner LA.;1; Noninvasive techinique for obtaining fluorescence excitation and emission spectra in vivo. Photochem Photobiol. 1986;44:679–87. [11] Ibbotson SH, Jong C, Lesar a., Ferguson JS, Padgett M, O’Dwyer M, et al.;1; Characteristics of 5-aminolaevulinic acid-induced protoporphyrin IX fluorescence in human skin in vivo. Photodermatol Photoimmunol Photomed. 2006;22(2):105–10. [12] Andrade CT, Vollet-Filho JD, Salvio AG, Bagnato VS, Kurachi C.;1; Identification of skin lesions through aminolaevulinic acid-mediated photodynamic detection. Photodiagnosis Photodyn Ther [Internet]. Elsevier B.V.; 2014;11(3):409–15. Available from: http://dx.doi.org/10.1016/j.pdpdt.2014.05.006 [13] Zeisser-Labouèbe M, Delie F, Gurny R, Lange N.;1; Screening of nanoparticulate delivery systems for the photodetection of cancer in a simple and cost-effective model. Nanomedicine (Lond). 2009;4(2):135–43. [14] Mondon K, Zeisser-Labouèbe M, Gurny R, Möller M.;1; MPEG-hexPLA micelles as novel carriers for hypericin, a fluorescent marker for use in cancer diagnostics. Photochem Photobiol. 2011;87(2):399–407. [15] Hammer-Wilson MJ, Akian L, Espinoza J, Kimel S, Berns MW.;1; Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for topically applied photosensitizers. J Photochem Photobiol B Biol. 1999;53(1-3):44–52. [16] Ruck a, Bohmler a, Steiner R.;1; PDT with TOOKAD (R) studied in the chorioallantoic membrane of fertilized eggs. Photodiagnosis Photodyn Ther. 2005;2:79–90.

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[17] Buzzá HH, Silva L V., Moriyama LT, Bagnato VS, Kurachi C;1;. Evaluation of vascular effect of Photodynamic Therapy in chorioallantoic membrane using different photosensitizers. J Photochem Photobiol B Biol. 2014;138:1–7. [18] Honda N, Kariyama Y, Hazama H, Ishii T, Kitajima Y, Inoue K, et al.;1; Optical properties of tumor tissues grown on the chorioallantoic membrane of chicken eggs: tumor model to assay of tumor response to photodynamic therapy. J Biomed Opt [Internet]. 2015;20(12):125001. Available from: http://biomedicaloptics.spiedigitallibrary.org/article.aspx?doi=10.1117/1. JBO.20.12.125001 [19] Yoon JH, Yoon HE, Kim O, Kim SK, Ahn SG, Kang KW.;1; The enhanced anti-cancer effect of hexenyl ester of 5-aminolaevulinic acid photodynamic therapy in adriamycinresistant compared to non-resistant breast cancer cells. Lasers Surg Med. 2012;44(1):76–86. [20] Fecchio D, Sirois P, Russo M, Jancar S;1; Studies on inflammatory response induced by Ehrlich tumor in mice peritoneal cavity. Inflammation [Internet]. 1990;14(1):125–32. Available from: http://www.ncbi.nlm.nih.gov/pubmed/2323805 [21] Buzza HH, Pires L, Bagnato VS, Kurachi C, Carlos S, Paulo S, et al.;1; Evaluation of the Photodynamic Therapy effect using a tumor model in Chorioallantoic Membrane with Melanoma Cells. In: Kessel DH, Hasan T, editors. Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XXII. 2014. p. 1–7. [22] Rodrigues PGS, Campos de Menezes PF, Fujita AKL, Escobar A, Barboza de Nardi A, Kurachi C, et al.;1; Assessment of ALA-induced PpIX production in porcine skin pretreated with microneedles. J Biophotonics. 2015 Sep;8(9):723–9. [23] de Andrade CT, Vollet-Filho JD, Salvio AG.;1; Identification of skin lesions through aminolevulinic acid-mediated photodynamic therapy. Photodiagnosis Photodyn Ther. 2014;11(3):409–4015. [24] Nissen CV, Philipsen P a., Wulf HC.;1; Protoporphyrin IX formation after topical application of methyl aminolaevulinate and BF-200 aminolaevulinic acid declines with age. Br J Dermatol [Internet]. 2015;n/a – n/a. Available from: http://doi.wiley.com/10.1111/bjd.13923 [25] Fritsch C, Lehmann P, Stahl W, Schulte KW, Blohm E, Lang K, et al.;1; Optimum porphyrin accumulation in epithelial skin tumours and psoriatic lesions after topical application of delta-aminolaevulinic acid. Br J Cancer. 1999;79(9-10):1603–8. [26] Robinson DJ, de Brujin HS, van der Veen N, Stringer MR, Brown SB, Star WM.;1; Photobleaching during and re-appearance after photodynamic therapy of topical ALA induced fluorescence in UVB-treated mouse skin. Photochem Photobiol. 1998;67:140–9.

Figure 1 - Experimental setup to obtain the fluorescence images of CAM.

Figure 2 - Monitoring of fluorescence of PpIX formation with ALA application over the time in the vessels of CAM model.

Figure 3 - Quantification of fluorescence ratio over time with ALA application (τ =8.3 ± 3.6 h) in the vessels of the CAM model. 10

Figure 4 - Monitoring of PpIX fluorescence in the vessels and tumor over time with ALA application. Arrows show the tumor region.

Figure 5 - Monitoring of Photogem® fluorescence over the time in the vessels of the CAM model.

Figure 6 - Quantification of fluorescence ratio over time with Photogem® application (τ = 2.8 ± 1.4 h) in the vessels of the CAM model.

Figure 7 - Monitoring of PpIX fluorescence with ALA application in the vessels and tumor with PDT. Illumination was applied after a 4-h incubation. Arrows indicate the tumor.

Figure 8 - Quantification of fluorescence of PpIX production with ALA application in the vessels and tumor before, during and after PDT. The red line corresponds to the moment of illumination. Table 1 Summary of the mean τ and R-Square found for both PS – PpIX and Photogem®

Blood vessels

PS

R-

Tumor Model

τ mean (h)

τ mean (h)

R-Square Square

CAM + Tumor

Δτ > 0

PpIX

8.3 ± 3.6

91±5 %

6.7 ± 2.5

2.1 ± 1.6

88±7 %

Photogem®

2.8 ± 1.4

87±4 %

4.5 ± 0.9

4.1 ± 1.4

79±9 %

TDENDOFDOCTD

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