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One important limitation of topical photodynamic therapy (PDT) is the limited tissue penetration of precursors. Microneedles (MNs) are minimally invasive devices used to promote intradermal drug delivery. Dissolving MNs contain drug-associated to polymer blends, dissolving after insertion into skin, allowing drug release. This study comprises development and characterization of a pyramidal model of dissolving MNs (500 μm) prepared with 5% wt/wt aminolevulinic acid and 20% wt/wt Gantrez AN-139 in aqueous blend. Protoporphyrin IX formation and distribution were evaluated in tumor mice model by using fluorescence widefield imaging, spectroscopy, and confocal microscopy. MNs demonstrated excellent mechanical resistance penetrating about 250 μm with minor size alteration in vitro, and fluorescence intensity was 5-times higher at 0.5 mm on average compared to cream in vivo (being 10 ± 5 a.u. for MNs and 2.4 ± 0.8 a.u. for cream). Dissolving MNs have overcome topical cream application, being extremely promising especially for thicker skin lesions treatment using PDT.

Purpose To investigate the safety of photobiomodulation therapy (PBM) in tumors and its potential as a radiosensitizer when combined with radiotherapy. Methods We have performed in vitro experiments in A431 cells to assess proliferation and cell cycle after PBM, as well as clonogenic assay and H2AX-gamma immunolabeling to quantify double strand breaks after the combination of PBM and radiation. In vivo experiments in xenografts included Kaplan-Meier survival analysis, optical coherence tomography (OCT) and histological analysis. Results PBM did not induce proliferation in vitro, but increased the G2/M fraction by 27% 24h after illumination, resulting in an enhancement of 30% in radiation effect in the clonogenic assay. The median survival of the PBM-RT group increased by 4 days and the hazard ratio was 0.417 (CI 95%: 0.173–1.006) when compared to radiation alone. OCT analysis over time demonstrated that PBM increases tumor necrosis due to radiation, and histological analysis showed that illumination increased cell differentiation and angiogenesis, which may play a role in the synergetic effect of PBM and radiation. Conclusion PBM technique may be one of the most appropriate approaches for radiosensitizing tumors while protecting normal tissue because of its low cost and low training requirements for staff.

Photodynamic therapy (PDT) is a technique with well-established principles that often demands repeated applications for sequential elimination of tumor cells. An important question concerns the way surviving cells from a treatment behave in the subsequent one. Threshold dose is a core concept in PDT dosimetry, as the minimum amount of energy to be delivered for cell destruction via PDT. Concepts of threshold distribution have shown to be an important tool for PDT results analysis in vitro. In this study, we used some of these concepts for demonstrating subsequent treatments with partial elimination of cells modify the distribution, which represents an increased resistance of the cells to the photodynamic action. HepG2 and HepaRG were used as models of tumor and normal liver cells and a protocol to induce resistance, consisted of repeated PDT sessions using Photogem® as a photosensitizer, was applied to the tumor ones. The response of these cells to PDT was assessed using a standard viability assay and the dose response curves were used for deriving the threshold distributions. The changes in the distribution revealed that the resistance protocol effectively eliminated the most sensitive cells. Nevertheless, HepaRG cell line was the most resistant one among the cells analyzed, which indicates a specificity in clinical applications that enables the use of high doses and drug concentrations with minimal damage to the surrounding normal tissue.

Graphene oxide (GO) with their interesting properties including thermal and electrical conductivity and antibacterial characteristics have many promising applications in medicine. The prevalence of resistant bacteria is considered a public health problem worldwide, herein, GO has been used as a broad spectrum selective antibacterial agent based on the photothermal therapy (PTT)/photodynamic therapy (PDT) effect. The preparation, characterization, determination of photophysical properties of two different sizes of GO is described. In vitro light dose and concentration-dependent studies were performed using Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria based on the PTT/PDT effect used ultra-low doses (65 mW cm–2) of 630 nm light, to achieve efficient bacterial decontamination. The results show that GO and nanographene oxide (nGO) can sensitize the formation of 1O2 and allow a temperature rise of 55°C to 60°C together nGO and GO to exert combined PTT/PDT effect in the disinfection of gram-positive S. aureus and gram-negative E. coli bacteria. A complete elimination of S. aureus and E. coli bacteria based on GO and nGO is obtained by using a dose of 43–47 J cm–2 for high concentration used in this study, and a dose of around 70 J cm–2 for low dose of GO and nGO. The presence of high concentrations of GO allows the bacterial population of S. aureus and E. coli to be more sensitive to the use of PDT/PTT and the efficiency of S. aureus and E. coli bacteria disinfection in the presence of GO is similar to that of nGO. In human neonatal dermal fibroblast, HDFs, no significant alteration to cell viability was promoted by GO, but in nGO is observed a mild damage in the HDFs cells independent of nGO concentration and light exposure. The unique properties of GO and nGO may be useful for the clinical treatment of disinfection of broad-spectrum antimicrobials. The antibacterial results of PTT and PDT using GO in gram-positive and gram-negative bacteria, using low dose light, allow us to conclude that GO and nGO can be used in dermatologic infections, since the effect on human dermal fibroblasts of this treatment is low compared to the antibacterial effect., GRAPHICAL ABSTRACT PTT/PTT effect due to irradiation with red LED of GO and nGO in the presence of E. coli and S. aureus bacteria.

In this chapter, we introduce the fundamentals of photodynamic reactions and applications for the treatment of oral-facial lesions, and microbiological control are presented.

Photobiomodulation therapy (PBMT) uses light to stimulate cells. The molecular basis of the effects of PBMT is being unveiled, but it is stated that the cytochrome-c oxidase enzyme in mitochondria, a photon acceptor of PBMT, contributes to an increase in ATP production and modulates the reduction and oxidation of electron carriers NADH and FAD. Since its effects are not fully understood, PBMT is not used on tumors. Thus, it is interesting to investigate if its effects correlate to mitochondrial metabolism and if so, how it could be linked to the optical redox ratio (ORR), defined as the ratio of FAD/(NADH + FAD) fluorescences. To that end, fibroblasts (HDFn cell line) and oral squamous cell carcinoma (SCC-25 cell line) were irradiated with a light source of 780 nm and a total dose of 5 J/cm2, and imaged by optical microscopy. PBMT down-regulated the SCC-25 ORR by 10%. Furthermore, PBMT led to an increase in ROS and ATP production in carcinoma cells after 4 h, while fibroblasts only had a modest ATP increase 6 h after irradiation. Cell lines did not show distinct cell cycle profiles, as both had an increase in G2/M cells. This study indicates that PBMT decreases the redox state of oral cancer by possibly increasing glycolysis and affects normal and tumor cells through distinct pathways. To our knowledge, this is the first study that investigated the effects of PBMT on mitochondrial metabolism from the initiation of the cascade to DNA replication. This is an essential step in the investigation of the mechanism of action of PBMT in an effort to avoid misinterpretations of a variety of combined protocols.

A combination of metabolic modifications by light stimulus and photodynamic action is very attractive. Photobiomodulation therapy (PBMT) comprehends a vast range of applications and has been shown to be suitable to ease morbidities caused by chemotherapy and radiation, such as mucositis and dermatitis. The current study investigates the effects of near-infrared PBMT combined with porphyrin-based photodynamic therapy (PDT) in squamous cell carcinoma cell lines SCC-25 and SCC-4. The aim is to evaluate the potential of this combination to improve PDT outcome by increasing cell toxicity. Many techniques were used to verify the combined effect. Photobiomodulation (PBM) enhanced PDT action in SCC-25 cells by increasing photosensitizer (PS) uptake and production of reactive oxygen species (ROS). The equivalent was not seen in SCC-4 cells compared to the PDT only group. We believe these effects are strongly related to the interval of application between PBMT, PS incubation and PDT. Additionally, the effect of ascorbic acid on preventing PBM effects in PDT shows that ROS play an important role in the early mechanisms of PBM-PDT. Therefore, we believe PBM-PDT combination is worth exploring, for its benefit-cost ratio and simple protocols, along with the possibility of improvement in treatment resuts.

Photodynamic therapy (PDT) is a technique with well-established principles that often demands repeated applications for sequential elimination of tumor cells. An important question concerns the way surviving cells from a treatment behave in the subsequent one. Threshold dose is a core concept in PDT dosimetry, as the minimum amount of energy to be delivered for cell destruction via PDT. Concepts of threshold distribution have shown to be an important tool for PDT results analysis in vitro. In this study, we used some of these concepts for demonstrating subsequent treatments with partial elimination of cells modify the distribution, which represents an increased resistance of the cells to the photodynamic action. HepG2 and HepaRG were used as models of tumor and normal liver cells and a protocol to induce resistance, consisted of repeated PDT sessions using Photogem® as a photosensitizer, was applied to the tumor ones. The response of these cells to PDT was assessed using a standard viability assay and the dose response curves were used for deriving the threshold distributions. The changes in the distribution revealed that the resistance protocol effectively eliminated the most sensitive cells. Nevertheless, HepaRG cell line was the most resistant one among the cells analyzed, which indicates a specificity in clinical applications that enables the use of high doses and drug concentrations with minimal damage to the surrounding normal tissue.

O princípio de terapia fotodinâmica (TFD) foi introduzido por volta de 1900 mas posteriormente investgado como candidato para tratamento de cancer na década de 1970. Desde então, existem diversos trabalhos a respeito do assunto in vitro, in vivo e em estudos clínicos e grandes avanços foram alcançados. Entretanto, alguns desafios ainda não foram superados, como a variabilidade dos resultados. Este trabalho consiste na investigação de suas causas, em que o principal objetivo é avançar o estado da arte em TFD. Para isso foi usado um modelo de distribuição de limiar de dose para avaliar resistência em TFD in vitro. As distribuições de limiar de dose são obtidas pela derivação a curva de dose resposta experimental. Elas são caracterizadas pela sua largura e pela dose que corresponde ao pico, que se relaciona a homogeneidade e resistência intrínseca da população, respectivamente. Na seção 1, é apresentada a avaliação e comparação de dados obtidos de resultados publicados na literatura e, na seção 2, de experimentos realizados pela autora em diferentes linhagens celulares. Da análise da primeira etapa, foi observado que a largura da distribuição é proporcional a dose do pico e foi possível investigar a dependência do resultado da TFD com a linhagem celular, dado um fotossensibilizador (FS). Foi interessante, também, notar que as distribuições de limiar de dose correspondem a curvas de atividade de marcadores celulares de apoptose, como função da dose de luz, para a maior parte das condições analisadas. Dos experimentos realizados pela autora, foi visto que as células normais são as mais resistentes ao dano, seguida das células de câncer resistentes e sua linhagem parental, e que sua resposta foi a mais homogênea. Essas observações foram corroboradas pelas imagens obtidas de microscopia de fluorescência para avaliação da captação de FS, que mostraram que as células tumorais acumulam mais FS que as outras. Portanto, foi mostrado o potencial de se aplicar distribuições de limiar de dose na análise de resultados de TFD in vitro, ela é uma poderosa ferramenta que fornece mais informações que as curvas de dose resposta padrão. The principle of photodynamic therapy (PDT) was introduced around 1900 but further investigated as a candidate to cancer treatment in the 1970´s. Since then, there are several papers regarding the subject in vitro, in vivo and clinical trials and great advances were achieved. However, some challenges were not yet overcome, such as results variability. This work consists in the investigation of its causes, where the main goal is to advance the state of art of PDT. For that it is being used a threshold dose distribution model to evaluate cell resistance to PDT in vitro. The threshold distributions are obtained by differentiating the experimental dose response curve. They are characterized by its width and the dose that corresponds to the peak which relates to the homogeneity and intrinsic resistance of the population, respectively. In section 1, it is presented the evaluation and comparison of data obtained from published results in literature and, in section 2, of experiments performed by the author in different cell lines. From the analysis in the first part, it was observed that the width of the distribution is proportional to its dose of the peak and it was possible to investigate the dependence of the PDT result with the cell line, given a fixed photosensitizer (PS). It was also interesting to note that the threshold distribution corresponded to the activity curves for apoptotic cell markers as a function of light dose, for most of the conditions analyzed. From the experiments performed by the author, it was seen that the normal cell line was the most resistant one, followed by the resistant cancer cells and its parental cell line, and that its response was more homogeneous. Those finding were supported by the fluorescence microscopy images obtained to evaluate PS uptake, which shown that the tumor cells accumulated more PS than the other ones. Therefore, it was shown the potential of applying the threshold distribution to analyze PDT results in vitro, it is a powerful tool that provides more information than the standard dose response curves.

The concept of threshold in photodynamic action on cells or microorganisms is well observed in experiments but not fully explored on in vitro experiments. The intercomparison between light and used photosensitizer among many experiments is also poorly evaluated. In this report, we present an analytical model that allows extracting from the survival rate experiments the data of the threshold dose distribution, ie, the distribution of energies and photosensitizer concentration necessary to produce death of cells. Then, we use this model to investigate photodynamic therapy (PDT) data previously published in literature. The concept of threshold dose distribution instead of "single value of threshold" is a rich concept for the comparison of photodynamic action in different situations, allowing analyses of its efficiency as well as determination of optimized conditions for PDT. We observed that, in general, as it becomes more difficult to kill a population, the distribution tends to broaden, which means it presents a large spectrum of threshold values within the same cell type population. From the distribution parameters (center peak and full width), we also observed a clear distinction among cell types regarding their response to PDT that can be quantified. Comparing data obtained from the same cell line and used photosensitizer (PS), where the only distinct condition was the light source's wavelength, we found that the differences on the distribution parameters were comparable to the differences on the PS absorption. At last, we observed evidence that the threshold dose distribution matches the curve of apoptotic activity for some PSs.