Induced photo-cytotoxicity on prostate cancer cells with the photodynamic action of toluidine Blue ortho.

BACKGROUND: Photodynamic therapy (PDT) has become an advantageous therapeutic approach for the treatment of select cancers and microbial infections. PDT generates toxic reactive oxygen species as an end product of the interaction between the photosensitizer and light with an appropriate wavelength. Toluidine blue ortho is a photosensitizer that is commonly used in the photodynamic treatment of bacterial infection and a promising photosensitizer for cancer treatment. This study aims to evaluate the potential photo-cytotoxicity of toluidine blue ortho-mediated photodynamic therapy on PC-3 prostate cancer cells. METHODS: In this study toluidine blue ortho-mediated photodynamic therapy was assessed on PC-3 cancer cells with various photosensitizer concentrations and light energy densities of the 655-nm diode laser. MTT analysis was used for the determination of the cytotoxicity on the cells and viability/cytotoxicity assay was used for live/dead cell staining after the applications. The mechanism of this application was further analyzed with the determination of intracellular reactive oxygen species and nitric oxide release. RESULTS: The light applications and the photosensitizer alone did not inhibit the cell viability of PC-3 cells. 20 J/cm2 laser energy density together with 100 µM photosensitizer concentration resulted in maximum cancer cell death with a rate of approximately 89 %. The level of intracellular reactive oxygen species increased with the increasing parameters of the applications that resulted in more cell death. CONCLUSION: This study showed the successful anticancer activity of toluidine blue ortho upon irradiation with 655 nm of laser light against PC-3 cancer cells and it was mediated with the production of reactive oxygen species.

Antibacterial activity of antimicrobial peptide-conjugated nanofibrous membranes.

Antimicrobial peptides (AMPs) are considered as novel potential alternatives to antibiotics against increasing number of multi drug resistant (MDR) pathogens. Although AMPs have shown strong antimicrobial activity against gram-negative or gram-positive microorganisms, AMP conjugated biomaterials that are effective against MDR microorganisms are yet to be developed. Herein, the potential use of (RWRWRWRW)-NH2 (AMP-1) and KRFRIRVRV-NH2 (AMP-2) peptide conjugated electrospun polylactic-co-glycolic-acid (PLGA) nanofibers (NFs) fabricated and their antimicrobial effect by themselves and in their dual combination (1:1) were evaluated on P. aeruginosa and methicillin-resistant S. aureus (MRSA). Those AMP conjugated NFs did not inhibit proliferation of keratinocytes. These results suggest that AMP conjugated NF, which has multiple biological activities, would be a promising candidate as a wound dressing material.

Effect of Integrin Binding Peptide on Vascularization of Scaffold-Free Microtissue Spheroids.

BACKGROUND: Three-dimensional (3D) biomimetic models via various approaches can be used by therapeutic applications of tissue engineering. Creating an optimal vascular microenvironment in 3D model that mimics the extracellular matrix (ECM) and providing an adequate blood supply for the survival of cell transplants are major challenge that need to be overcome in tissue regeneration. However, currently available scaffolds-depended approaches fail to mimic essential functions of natural ECM. Scaffold-free microtissues (SFMs) can successfully overcome some of the major challenges caused by scaffold biomaterials such as low cell viability and high cost. METHODS: Herein, we investigated the effect of soluble integrin binding peptide of arginine-glycine-aspartic acid (RGD) on vascularization of SFM spheroids of human umbilical vein endothelial cells. In vitro-fabricated microtissue spheroids were constructed and cultivated in 0 mM, 1 mM, 2 mM, and 4 mM of RGD peptide. The dimensions and viability of SFMs were measured. RESULTS: Maximum dimension and cell viability observed in 2 mM RGD containing SFM. Vascular gene expression of 2 mM RGD containing SFM were higher than other groups, while 4 mM RGD containing SFM expressed minimum vascularization related genes. Immunofluorescent staining results indicating that platelet/endothelial cell adhesion molecule and vascular endothelial growth factor protein expression of 2 mM RGD containing SFM was higher compared to other groups. CONCLUSION: Collectively, these findings demonstrate that SFM spheroids can be successfully vascularized in determined concentration of RGD peptide containing media. Also, soluble RGD incorporated SFMs can be used as an optimal environment for successful prevascularization strategies.

Enhanced osteogenesis of human mesenchymal stem cells by self-assembled peptide hydrogel functionalized with glutamic acid templated peptides.

Self-assembling peptide (SAP) hydrogel has been shown to be an excellent biological material for three-dimensional cell culture and stimulatie cell migration and differentiation into the scaffold, as well as for repairing bone tissue defects. Herein, we designed one of the SAP scaffolds KLD (KLDLKLDLKLDL) through direct coupling to short bioactive motif O1 (EEGGC) and O2 (EEEEE) of which bioactivity on osteogenic differentiation was previously demonstrated and self-assembled in different concentrations (0.5%, 1%, and 2%). Our aim was to enhance osteogenesis and biomineralization of injectable SAP hydrogels with controlled mechanical properties so that the peptide hydrogel also becomes capable of being injected to bone defects. The molecular integration of the nanofibrous peptide scaffolds was observed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The rheological properties and degradation profile of SAP hydrogels were evaluated to ensure stability of SAPs. Compared with pure KLD scaffold, we found that these designed bioactive peptide scaffolds significantly promoted hMSCs proliferation depicted by biochemical analysis of alkaline phosphatase (ALP) activity, total calcium deposition. Moreover, key osteogenic markers of ALP activity, collagen type I (COL-1), osteopontin (OP), and osteocalcin (OCN) expression levels determined by real-time polymerase chain reaction (PCR) and immunofluorescence analysis were also significantly increased with the addition of glutamic acid residues to KLD. We demonstrated that the designed SAP scaffolds promoted the proliferation and osteogenic differentiation of hMSCs. Our results suggest that these designed bioactive peptide scaffolds may be useful for promoting bone tissue regeneration.

The effect of indocyanine green-based photodynamic therapy on healthy fibroblast and keratinocyte cells.

BACKGROUND: Photodynamic therapy is a promising invention to treat infections and cancer where conventional treatments are insufficient and have many side effects. Photodynamic therapy is mainly emphasized as having minimal side effects on healthy cells during local applications, even so photosensitizer can accumulate in any cell and unwanted deaths may occur upon irradiation. This study focused on the degree of photodynamic action with indocyanine green against healthy cells, when it has phototoxic effects on pathogens. METHODS: Healthy mouse skin fibroblast and human skin keratinocyte cells were exposed to energy densities of 84 and 252 J/cm2 with 4, 10, 25, 50,100, 125 and 150 µg/mL indocyanine green which have efficiently killed gram-positive and gram-negative pathogens. Cell Viability, Lipid Peroxidation and Live/Dead Cell Staining analysis were performed to assess the phototoxicity with defined parameters on the healthy cells. RESULTS: 84 J/cm2 energy density was quite safe for keratinocytes with indocyanine green concentrations ranging from 4 to 125 µg/mL. When 252 J/cm2 energy density was used, most of the keratinocytes were damaged with any photosensitizer concentration. Fibroblasts only tolerate these energy densities together with 4 and 10 µg/mL indocyanine green. Increasing photosensitizer concentrations resulted in high phototoxic effect on them. CONCLUSION: Photodynamic therapy applications, which destroy pathogens, may also kill healthy eukaryotic cells. While some energy densities are safe, but others cause serious mortality rate on fibroblasts and keratinocytes. Therefore, harm to healthy cells related to photodynamic therapy parameters should be minimized by the optimization of energy densities and photosensitizer concentration properly.

Guiding neural extensions of PC12 cells on carbon nanotube tracks dielectrophoretically formed in poly(ethylene glycol) dimethacrylate.

The PC12 cell line has been widely used as an in vitro model for studying neuronal differentiation and identifying the factors affecting the process. It has the ability to differentiate in the presence of nerve growth factor (NGF), resulting in neural extensions called dendrites and axons. In this study, first the impact of randomly distributed multi-walled carbon nanotubes (MWCNTs) in poly(ethylene glycol) dimethacrylate (PEGDMA) on PC12 cell differentiation was investigated in terms of neurite length, number of neurite per cell and differentiation marker gene expression profile. Then, dielectrophoretically aligned MWCNTs in PEGDMA was used to guide and support the neuronal differentiation of PC12 cells in the presence of NGF. The method is expected to be useful in revealing the nanotopographical role in fundamental studies and understanding of nanotopographical effects for biomedical applications on nerve regeneration.

Accelerated mineralization on nanofibers via non-thermal atmospheric plasma assisted glutamic acid templated peptide conjugation.

Surface modification by non-thermal atmospheric plasma (NTAP) treatment can produce significantly higher carboxylic groups on the nanofibers (NF) surface, which potentially can increase biomineralization of NF via promoting glutamic acid (GLU) templated peptide conjugation. Herein, electrospun poly(lactide-co-glycolide) (PLGA) scaffolds were treated with NTAP and conjugated with GLU peptide followed by incubation in simulated body fluids for mineralization. The effect of NTAP treatment and GLU peptide conjugation on mineralization, surface wettability and roughness were investigated. The results showed that NTAP treatment significantly increased GLU peptide conjugation which consequently enhanced mineralization and mechanical properties of NTAP treated and peptide conjugated NF (GLU-pNF) compared to neat PLGA NF, NTAP treated NF (pNF) and GLU peptide conjugated NF (GLU-NF). The effect of surface modification on human bone marrow derived mesenchymal stem cells adhesion, proliferation and morphology was evaluated by cell proliferation assay and fluorescent microscopy. Results demonstrated that cellular adhesion and proliferation were significantly higher on GLU-pNF compared to NF, pNF and GLU-NF. In summary, NTAP treatment could be a promising modification technique to induce biomimetic peptide conjugation and biomineralization for bone tissue engineering applications.

Aspartic and Glutamic Acid Templated Peptides Conjugation on Plasma Modified Nanofibers for Osteogenic Differentiation of Human Mesenchymal Stem Cells: A Comparative Study.

Optimization of nanofiber (NF) surface properties is critical to achieve an adequate cellular response. Here, the impact of conjugation of biomimetic aspartic acid (ASP) and glutamic acid (GLU) templated peptides with poly(lactic-co-glycolic acid) (PLGA) electrospun NF on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Cold atmospheric plasma (CAP) was used to functionalize the NF surface and thus to mediate the conjugation. The influence of the CAP treatment following with peptide conjugation to the NF surface was assessed using water contact angle measurements, Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The effect of CAP treatment on morphology of NF was also checked using Scanning Electron Microscopy (SEM). Both the hydrophilicity of NF and the number of the carboxyl (-COOH) groups on the surface increased with respect to CAP treatment. Results demonstrated that CAP treatment significantly enhanced peptide conjugation on the surface of NF. Osteogenic differentiation results indicated that conjugating of biomimetic ASP templated peptides sharply increased alkaline phosphatase (ALP) activity, calcium content, and expression of key osteogenic markers of collagen type I (Col-I), osteocalcin (OC), and osteopontin (OP) compared to GLU conjugated (GLU-pNF) and CAP treated NF (pNF). It was further depicted that ASP sequences are the major fragments that influence the mineralization and osteogenic differentiation in non-collagenous proteins of bone extracellular matrix.