Most of the nanoimaging tools like quantum dots and metallic nanoparticles are shown to have different levels of cytotoxicity via various mechanisms. However carbon nanodots (CNDs) are a new group of ultra small nano structures (average 4–6 nm) which is potential candidate of next generation optical imaging. Being carbonaceous in origin, CNDs possess excellent luminescence and photostability with significantly less cytotoxicity. In present study, we have synthesized carbon nano-dots from date molasses by microwave irradiation at ∼pH 11. The synthesized carbon nanodots were characterized using UV-Vis spectroscopy, fluorescence spectroscopy, TEM, XRD analysis, FTIR study and Zeta potential measurement. The average sizes of the dots were found to be 5–7 nm. A clear band emission was visible around 480 nm when an excitation beam of 415 nm was incident. For biological applicability, MTT assay and hemocompatibility studies were performed. The results exhibited the material to be highly cytocompatible within the application limit. Upon immediate exposure to CNDs, no significant changes to cellular surface morphology were observed via AFM imaging. Significant hemolysis or blood cell aggregation was not observed after incubation of CNDs with blood. After labelling with CNDs, MG-63 cells were found to be unbleached up to several hours even on exposure to light. We are reporting first time in this study the free radical scavenging property of CNDs in ex vivo and in vitro models. Antioxidant activity was measured ex vivo via potassium permanganate assay and DPPH assay. In vitro superoxide inhibition activity was measured both by spectroscopy and under microscope by NBT reduction assay. Hydroxyl free radical inhibition activity was measured via DCFH-DA Assay. The results were comparable with scavenging activity of standard antioxidant molecules (BHT and L-ascorbic acid). A novel assay for quantitative analysis of cellular oxidative stress was also proposed. Therefore, this material could be useful for long-term live cell imaging and cell tracking in a scaffold with minimal cytotoxicity and oxidative stress.

Mimicking skin extracellular matrix hierarchy, the present work aims to develop a bilayer skin graft comprising a porous cotton-wool-like 3D layer with membranous structure of PCL–chitosan nanofibers. Emulsion electrospinning with differential stirring periods of PCL–chitosan emulsion results in development of a bilayer 3D structure with varied morphology. The electrospun membrane has fiber diameter ∼274 nm and pore size ∼1.16 μm while fluffy 3D layer has fiber diameter ∼1.62 μm and pore size ∼62 μm. The 3D layer was further coated with collagen I isolated from Cirrhinus cirrhosus fish scales to improve biofunctionality. Surface coating with collagen I resulted in bundling the fibers together, thereby increasing their average diameter to 2.80 μm and decreasing pore size to ∼45 μm. The architecture and composition of the scaffold promotes efficient cellular activity where interconnected porosity with ECM resembling collagen I coating assists cellular adhesion, infiltration, and proliferation from initial days of fibroblast seeding, while keratinocytes migrate on the surface only without infiltrating in the membranous nanofiber layer. Anatomy of the scaffold arising due to variation in pore size distribution at different layers thereby facilitates compartmentalization and prevents initial cellular transmigration. The scaffold also assists in extracellular matrix protein synthesis and keratinocyte stratification in vitro. Further, the scaffold effectively integrates and attaches with third-degree burn wound margins created in rat models and accelerates healing in comparison to standard Tegaderm dressing™. The bilayer scaffold is thus a promising, readily available, cost-effective, off-the-shelf matrix as a skin substitute.