Since Graphene discovery, their associated derivate nanomaterials, Graphene Oxide (GO) and reduced-GO were in the forefront of continuous developments in bio-nano-technology due to unique physical-chemical properties. Although GO nano-colloids (GON) were proposed as drug release matrix for targeting cancer cells, there is still a concern regarding its cytotoxicity issues. In this study, we report on the fabrication of functional GON bio-coatings by Matrix-Assisted Pulsed Laser Evaporation (MAPLE) to be used as drug carriers for targeting melanoma cells. We first performed a thorough in vitro cytotoxicity assay for comparison between GON and protein functionalized GON coatings. As functionalization protein, Bovine Serum Albumin (BSA) was non-covalently conjugated to GO surface. Safe concentration windows were identified in cytotoxicity tests by live/dead staining and MTS assays for five different human melanoma cell lines as well as for non-transformed melanocytes and human dermal fibroblasts. Hybrid GON-BSA nano-scaled thin coatings incorporating Dabrafenib (DAB) and Trichostatin A (TSA) inhibitors for cells bearing BRAF V600E pathway activating mutation were assembled on solid substrates by MAPLE technique. We further demonstrated the successful immobilization for each drug-containing GON-BSA assembling systems by evaluating cellular BRAF activity inhibition and histone deacetylases activity blocking, respectively. DAB activity was proven by the decreased ERK phosphorylation in primary melanoma cells (SKmel28 BRAF V600E cell line), while TSA effect was evidenced by acetylated histones accumulation in cell's nuclei (SKmel23 BRAF WT cell line). In addition, melanoma cells exposed to GON-BSA coatings with compositional gradient of inhibitors evidenced a dose-dependent effect on target activity. Such functional bio-platforms could present high potential for cell-biomaterial interface engineering to be applied in personalized cancer therapy studies.
In this study, we report on the synthesis of L-Cysteine (L-Cys)-coated magnetic iron oxide nanoparticles (NPs) loaded with doxorubicin (Dox). The Fe3O4-L-Cys-Dox NPs were extensively characterized for their compositional and morpho-structural features using EDS, SAED, XRD, FTIR and TEM. XPS, Mӧssbauer spectroscopy and SQUID measurements were also performed to determine the electronic and magnetic properties of the Fe3O4-L-Cys-Dox nanoparticles. Moreover, by means of a FO-SPR sensor, we evidenced and confirmed the binding of Dox to L-Cys. Biological tests on mouse (B16F10) and human (A375) metastatic melanoma cells evidenced the internalization of magnetic nanoparticles delivering Dox. Half maximum inhibitory concentration IC50 values of Fe3O4-L-Cys-Dox were determined for both cell lines: 4.26 µg/mL for A375 and 2.74 µg/mL for B16F10, as compared to 60.74 and 98.75 µg/mL, respectively, for unloaded controls. Incubation of cells with Fe3O4-L-Cys-Dox modulated MAPK signaling pathway activity 3 h post-treatment and produced cell cycle arrest and increased apoptosis by 48 h. We show that within the first 2 h of incubation in physiological (pH = 7.4) media, ~10–15 µM Dox/h was released from a 200 µg/mL Fe3O4-L-Cys-Dox solution, as compared to double upon incubation in citrate solution (pH = 3), which resembles acidic environment conditions. Our results highlight the potential of Fe3O4-L-Cys-Dox NPs as efficient drug delivery vehicles in melanoma therapy.
Various material processing techniques have been proposed for fabrication of smart surfaces that can modulate cellular behavior and address specific clinical issues. Among them, laser-based technologies have attracted growing interest due to processing versatility. Latest development of ultrashort pulse lasers with pulse widths from several tens of femtoseconds (fs) to several picoseconds (ps) allows clean microfabrication of a variety of materials at micro- and nanoscale both at surface and in volume. In this study, we addressed the possibility of 3D microfabrication of photosensitive glass (PG) by high repetition rate ps laser-assisted etching (PLAE) to improve the fabrication efficiency for the development of useful tools to be used for specific biological applications. Microfluidic structures fabricated by PLAE should provide the flow aspects, 3D characteristics, and possibility of producing functional structures to achieve the biologically relevant microenvironments. Specifically, the microfluidic structures could induce cellular chemotaxis over extended periods in diffusion-based gradient media. More importantly, the 3D characteristics could reproduce capillaries for in vitro testing of relevant organ models. Single cell trapping and analysis by using the fabricated microfluidic structures are also essential for understanding individual cell behavior within the same population. To this end, this paper demonstrates: (1) generation of 3D structures in glass volume or on surface for fabrication of microfluidic channels, (2) subtractive 3D surface patterning to create patterned molds in a controlled manor for casting polydimethylsiloxane (PDMS) structures and developing single cell microchambers, and (3) designing glass photo-masks to be used for sequel additive patterning of biocompatible nanomaterials with controlled shapes, sizes, and periodicity. Mesenchymal stem cells grown on laser-processed glass surfaces revealed no sign of cytotoxicity, while a collagen thin coating improved cellular adhesion.
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