CC BY
12DOI 10.24412/cl-37136-2023-1-176-180
STRATEGIES TO IMPROVE TOPICAL PHOTODYNAMIC THERAPY EFFICACY USING
ANIMAL MODELS
MIRIAN DENISE STRINGASCI1 AND VANDERLEI SALVADOR BAGNATO1,2
1Sao Carlos Institute of Physics, University of Sao Paulo, Brazil 2Department of Biomedical
Engineering, Texas A&M University, USA
ABSTRACT
Photodynamic Therapy (PDT) is a therapeutic option for the treatment of malignant and potentially malignant lesions. This technique is based on a photosensitizer (PS) excited with light at a suitable wavelength for absorption. The interaction of excited PS with molecular oxygen generates reactive oxygen species that lead to cell death. [1] One of the limiting factors of topical PDT is the cutaneous permeation of the PS or precursor, as in the case of aminolevulinic acid (ALA) and methyl aminolevulinate (Methyl-ALA). There are reports that there is a strong relationship between temperature and the synthesis of porphyrin IX (PpIX) in biological tissue. This suggests that increasing skin temperature during ALA or Methyl-ALA incubation may increase tissue accumulation of PpIX, improving the effectiveness of PDT, especially in areas that naturally experience lower temperatures, such as the extremities. [2, 3] In a skin model, Wistar rats were used with the administration of cream containing ALA or Methyl-ALA, both at a concentration of 20%, for 15 minutes. The animals were divided into 5 groups: tissue cooling to 20 °C or tissue heating to 40 °C (before or after cream incubation) and the control group (unchanged temperature). In each animal, two regions of the back were shaved and each group was composed of 4 treatment regions. From collections of fluorescence spectra, using a laser for excitation at 408 nm, it was possible to evaluate the PpIX emission signal at 630 nm, which is directly related to the accumulation of the molecule in the tissue. [4] It was found that both ALA and Methyl-ALA showed increased PpIX production when tissue was heated before cream incubation, showing better penetration (Fig. 1). When the temperature was changed after incubation of the cream, PpIX production decreased by both heating and cooling, probably because the enzymes that make up biological tissue were no longer at their optimal functioning, which occurs at normal tissue temperature (around of 37°C), Fig. 2.
initial Omin 60min 120min 180min initial Omin 60min 120min 180min
after the temperature change after the temperature change
Figure 1: Variation of PpIX production due to skin temperature change for 15 minutes before cream application (A) comparing the regions with methyl application; (B) ALA application; (C) comparing ALA and methyl without temperature change; (D) comparing ALA and methyl in tissue previously cooled to 20 ° C and (E) comparing ALA and methyl in previously heated tissue at 40 ° C
Figure 2: Variation of PpIX production due to skin temperature change for 180 minutes after cream application (A) comparing the regions with methyl application; (B) ALA application; (C) comparing ALA and methyl without temperature change; (D) comparing ALA and methyl in tissue subsequently cooled to 20 ° C and (E) comparing ALA and methyl in subsequently heated tissue at 40 ° C.
Another study aimed to test thermogenic/vasodilating agents incorporated into the precursor creams ALA and MethylALA to assess a possible improvement in the production of PpIX, in which menthol, methyl nicotinate (MN), and the extract of ginger. Again, healthy skins of Wistar rats were used, with 4 regions treated in each group, in the control group ALA or Methyl-ALA creams were used without the addition of agents. In each animal, two regions of the back were shaved, the cream containing 20% ALA was applied to one region, and the cream containing 20% Methyl-ALA was applied to the other. The creams were maintained with an occlusive dressing for 3 hours. Fluorescence spectroscopy measurements were used to assess PpIX formation, the results are presented in Fig. 3. [5]
Figure 3: (A) Comparison of the PpIX production of PpIX from the different agents incorporated into the
cream containing ALA and (B) and methyl-ALA.
It is possible to observe in Fig. 3 (B) that MN associated with Methyl-ALA showed the highest production of PpIX, at the end of 180 minutes this production is about 50% greater than that observed by Methyl-ALA without an added agent. The use of this agent may present a possible strategy to reduce the incubation time and/or increase PpIX production in more keratinized lesions. A new study was carried out to evaluate this better formulation (0.5% MN incorporated into 20% Methyl-ALA), in cell culture, non-cytotoxicity and increased cell metabolism were observed using confocal microscopy to calculate the redox rate. It was also possible to note the vasodilator effect of MN in a chorioallantoic membrane model (Fig. 4), in which after incubation with a 1% MN solution, the vessels dilated enough to rupture the smaller vessels and cause a small hemorrhage. in the demarcated region.
Figure 4: Images of vessels in the chorioallantoic membrane model (A)before and (B) after sixty minutes
of incubation with 1% MN.
To evaluate the damage caused by the accumulation of PpIX in each situation, we performed PDT with the illumination using the LINCE® equipment (LINCE, MMOptics, Brazil) at the wavelength of 630 nm, with an intensity of 125 mW/cm2 for 20 min, totaling a dose of 150 J/cm2 . In Fig. 5 the necrosis generated in the tissues three days after the treatments using different incubation times of the cream Methyl-ALA with incorporated MN can be observed.
F •» (B)
•i M V > >7 1 ..
(D) ^^^
Figure 5: Necrosis by PDT using (A) Methyl-ALA and MN for 1 hour; (B) Methyl-ALA and MN for 2 hours; (C) Methyl-ALA and MN for 3 hours and (D) pure Methyl-ALA for 3 hours.
The analysis of the photographic images of damages and the histological slides showed that the incorporation of MN with Methyl-ALA increased the damage induced in the epidermis after PDT. Another important factor to ensure the effectiveness of PDT is the adequate presence of O2 molecules in the tissue. The PDT procedure is highly O2 dependent and its effectiveness is affected when there is a dramatic consumption of O2. Thus, to define the best protocol, the irradiance must consider the tissue oxygenation rate via blood flow to
ensure greater efficiency in the production of reactive oxygen species (ROS). [6-8] In a new study, vascular and tissue damages were evaluated for different PDT protocols applied in squamous cell carcinoma implanted in balb/c nude mice. The administration of PS occurred topically or systemically. For topical applications, about 30 ^L of cream containing 20% MAL (PDT Pharma®, Cravinhos, Brazil) was positioned over the tumor, and incubated for one hour to accumulate PplX. For systemic application, Photogem (Photogem®, Moscow, Russia) solution was prepared at 1.5 mg/kg of the animal's body mass concentration, diluted in saline, and injected intraperitoneally with an incubation time of 6 hours. All animals were irradiated using LINCE® equipment (MMOptics, Sao Carlos, Brazil) that is composed of LEDs emitting at 630 nm. The fluence deposited was 60 J/cm2 , varying in sort irradiation (100 mW/cm2 for 10 min) or long irradiation (50 mW/cm2 for 20 min). Angiographic images in and around the tumor region were recorded by optical coherence tomography (OCT) using the Telesto 320C model (ThorLab®, USA). Vessel density in the images was evaluated using ImageJ Software for the different protocols. It is possible to observe greater vascular destruction (lower vessel density) in tumors treated with topical photosensitization and short irradiation (Fig. 6a).
Figure 6: Post PDT vessel density for different protocols assessed (a) in the tumor and (b) around the
tumor.
It is also possible to observe that both in the tumor and around it (Fig. 6a and b, respectively), the treatments with shorter irradiation periods and higher irradiance (all the protocols were set with the same fluence) presented a greater capacity to eliminate vessels, probably by causing higher temperature rise and shorter time for cell repair. Understanding tissue vascularization after treatment is essential for planning new protocols, especially in cases of treatments that require more than one session. The results of these studies are promising for the development of more effective topical PDT protocols for the treatment of non-melanoma skin lesions.
REFERENCES
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