Replicating endothelial shear stress in organ-on-a-chip for predictive hypericin photodynamic efficiency.

Photodynamic therapy using Hypericin (Hy-PDT) is an alternative non-invasive treatment that enables selective tumor inhibition and angiogenesis derived from the differential recruitment of endothelial cells in the tumor microenvironment. Most PDT studies were performed on in vitro models without vascular biomechanical simulation. Our work strives to develop a microchip that generates a constant shear stress force to investigate the Hy-PDT efficiency on human umbilical vein endothelial cells (HUVECs). The microchip with a single straight microchannel was composed of the bottom layer (polystyrene), the middle layer (double-sided biocompatible adhesive tape), and the top layer (polyester film) and could produce shear stress in the range of 1.4 - 7.0 dyn cm-2. The quantification of vascular endothelial growth factor (VEGF), cell viability, and activities of caspases 3 and 7 were assayed to validate the microchip and Hy-PDT efficacy. After the endothelization, static and dynamic cell incubations with Hy were conducted in microchips. Compared to static systems, the shear stress displayed its effect on the increasing release of VEGF and promoted more cell damage and cell death via necrosis during Hy-PDT. In conclusion, the expressive shear stress-dependent manner during PDT treatments suggests that the microchip could be an essential approach in preclinical tests to evaluate the therapeutic outcome considering the endothelial shear stress microenvironment.

Rapid Fabrication of Microfluidic Devices for Biological Mimicking: A Survey of Materials and Biocompatibility.

Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept.

Evidence of hypericin photoinactivation of E. faecalis: From planktonic culture to mammalian cells selectivity up to biofilm disruption.

Antimicrobial Photodynamic Therapy (aPDT) is an alternative for microbiological inactivation. The aPDT is a method that uses a photosensitizer (PS) excited by visible light at the appropriate wavelength and the molecular oxygen present in the tissues resulting in the production of reactive oxygen species, which causes oxidative damage to biological molecules. This study aimed to perform an in vitro experimental sequence for photoinactivation of E. faecalis using Hypericin (HY) from planktonic culture to selectivity assays using mammalian cells up to biofilm. The results show that E. faecalis rapidly absorb HY. The levels of inactivation of E. faecalis reached up to 99% in planktonic culture. Transmission and Scanning Electron Microscopy demonstrate the remarkable morphological alterations resulting from photooxidation being the loss of membrane integrity assessed by fluorescence microscopy combined with a LIVE/DEAD™ kit. HY did not present cytotoxicity to the fibroblasts cell at the used conditions proving to be a selective molecule. Finally, 60% of photoinactivation was observed in the biofilm of E. faecalis when subject to HY-aPDT. These outcomes show the advantages of sequential in vitro experiments besides showing that HY is a potential PS for clinical trials due to its selectivity and photodynamic effect. This study also draws attention to the benefits of using methodologies that can evidence the antimicrobial effect beyond the typical constellation of cell death.