Development of 3D skin equivalents for application in photodynamic biostimulation therapy assays using curcumin nanocapsules.

For decades, animal models have been the standard approach in drug research and development, as they are required by regulations in the transition from preclinical to clinical trials. However, there is growing ethical and scientific concern regarding these trials, as 80 % of the therapeutic potential observed in pre-clinical studies are often unable to be replicated, despite demonstrating efficacy and safety. In response to this, Tissue Engineering has emerged as a promising alternative that enables the treatment of various diseases through the production of biological models for advanced biological assays or through the direct development of tissue repairs or replacements. One of the promising applications of Tissue Engineering is the development of three-dimensional (3D) models for in vitro tests, replacing the need for in vivo animal models. In this study, 3D skin equivalents (TSE) were produced and used as an in vitro model to test photobiostimulation using curcumin-loaded nanocapsules. Photodynamic biostimulation therapy uses photodynamic processes to generate small amounts of reactive oxygen species (ROS), which can activate important biological effects such as cell differentiation, modulation of inflammatory processes and contribution to cell regeneration. The PLGA nanocapsules (NC) used in the study were synthesized through a preformed polymer deposition method, exhibiting particle size <200 nm, Zeta potential >|30| and polydispersity index between 0.5 and 0.3. Atomic force microscopy analyzes confirmed that the particle size was <200 nm, with a spherical morphology and a predominantly smooth and uniform surface. The NC biocompatibility assay did not demonstrate cytotoxicity for the concentrations tested (2.5-25 µg mL-1).The in vitro release assay showed a slow and sustained release characteristic of the nanocapsules, and cellular uptake assays indicated a significant increase in cellular internalization of the curcumin-loaded nanostructure. Monolayer photobiostimulation studies revealed an increase in cell viability of the HDFn cell line (viability 134 %-228 %) for all LED fluences employed at λ = 450 nm (150, 300, and 450 mJ cm-2). Additionally, the scratch assays, monitoring in vitro scar injury, demonstrated more effective effects on cell proliferation with the fluence of 300 mJ cm-2. Staining of TSE with hematoxylin and eosin showed the presence of cells with different morphologies, confirming the presence of fibroblasts and keratinocytes. Immunohistochemistry using KI-67 revealed the presence of proliferating cells in TSE after irradiation with LED λ = 450 nm (150, 300, and 450 mJ cm-2).

Enzymatic production process of capric acid-rich structured lipids: Development of formulation as a new therapeutic approach.

The present work reports an optimization of the synthesis of MLM-type (medium, long, medium) structured lipids (SL) through an acidolysis reaction of grape seed oil with capric acid catalyzed by Rhizopus oryzae lipase immobilized. At first, tests were carried out by preparing the biocatalysts using enzyme loadings (0.15 to 1 g of enzymatic powder) for each gram of support. Enzyme loading was used 0.3 g of enzymatic powder, and hydrolytic activity of 1860 ± 23.4 IU/g was reached. Optimized conditions determined by the Central Composite Rotatable Design (CCRD) revealed that the acidolysis reaction reached approximately 59 % incorporation degree (%ID) after 24 h, in addition to the fact that the biocatalyst could maintain the incorporation degree in five consecutive cycles. From this high incorporation degree, cell viability assays were performed with murine fibroblast cell lines and human cervical adenocarcinoma cell lines. Concerning the cytotoxicity assays, the concentration of MLM-SL to 1.75 and 2 % v/v were able to induce cell death in 56 % and 64 % of adenocarcinoma cells, respectively. Human cervical adenocarcinoma cells showed greater sensitivity to the induction of cell death when using emulsions with MLM-SL > 1.75 % v/v compared to emulsions with lower content indicating a potential for combating carcinogenic cells.

Advanced methylene blue - nanoemulsions for in vitro photodynamic therapy on oral and cervical human carcinoma.

Photodynamic therapy (PDT) is a therapeutic modality with high contributions in the treatment of cancer. This approach is based on photophysical principles, which presents as a less invasive strategy than conventional therapies. Combined with nanotechnology, the therapy becomes more efficient because nanoparticles (NPs) have advantageous characteristics such as biocompatibility, controlled, and targeted release, promoting solubility and decreasing the toxicity and side effects involved. In this work were developed nanoemulsions containing the methylene blue photosensitizer (MB) (MB/NE) and in the empty form (unloaded/NE). Subsequently, the mentioned nanomaterials were characterized by the measurement of dynamic light scattering (DLS). The MB/NE and unloaded/NE showed appropriate physical and chemical characteristics, with particle size ≤ 200 nm, polydispersity index close to 0.3, and zeta potential exhibiting negative charge, showing stable values during the analysis. The incorporation of the MB did not cause changes in the photophysical profile of the photosensitizer. The quantification performed showed an incorporation rate of 81.9%. Viability studies showed an absence of cytotoxicity for MB/NE in the concentrations of 10-75 µmol·L-1, free MB at the concentration of 75 µmol·L-1, and unloaded NE 47.5% (v/v), presenting viability close to 90%, respectively. PDT in vitro protocols applied to OSCC and HeLa cells showed a decrease in cell viability through only one irradiation, evidencing the photodynamic activity of the formulation when applied to cancer cells. The results obtained were superior to those found in the literature where they use free MB, showing that the association between nanotechnology and PDT optimizes the proposed protocol. From the results obtained, it is possible to indicate that the NE have high stability, with satisfactory physical-chemical parameters, in addition to not presenting cytotoxicity in the tested concentrations, showing their in vitro biocompatibility, in addition to presenting satisfactory effects when combined MB/NE with PDT, showing the potential of MB/NE as a very promising nanostructured photosensitizer for the treatment of some types of cancer.