PURPOSE.To model the photochemical kinetics of corneal crosslinking with riboflavin (Rf) and confirm the model through measured oxygen concentration experiments under varying energy input conditions by UV-A irradiance and temperature modulation in ex vivo porcine cornea.
METHODS.A theoretical model was developed to describe the corneal cross-linking photochemical kinetics of Rf. After instillation with drops of Rf solution in distilled water, deepithelialized porcine corneas were exposed to 365-nm ultraviolet light (UV-A) under varying irradiance and temperature. Oxygen concentration in the cornea at a known depth was monitored during UV-A illumination with a dissolved oxygen fiberoptic microsensor. Data from the oxygen experiments were used to confirm the model.
RESULTS.On the basis of the known chemical reactions and diffusion rates of Rf and oxygen into the cornea, the authors developed a theoretical model consistent with corneal oxygen consumption experimental results during UV-A irradiation under different conditions. Oxygen concentration in the cornea is modulated by UV-A irradiance and temperature and quickly decreased at the beginning of UV-A exposure. The time-dependence of both Type-I and Type-II photochemical mechanisms in corneal cross-linking with Rf are discussed.CONCLUSIONS. Using a chemical kinetics modeling approach, the authors developed a simple model that is in agreement with their experimental results on oxygen consumption in the cornea during corneal cross-linking with Rf. It is suggested that the main photochemical kinetics mechanism is the direct interaction between Rf triplets and reactive groups of corneal proteins, which leads to the cross-linking of the proteins mainly through radical reactions. (Invest Ophthalmol Vis Sci.
This study tested the hypothesis that controlled flow through microchannels can cause shear-induced intracellular loading of cells with molecules. The overall goal was to design a simple device to expose cells to fluid shear stress and thereby increase plasma membrane permeability. DU145 prostate cancer cells were exposed to fluid shear stress in the presence of fluorescent cell-impermeant molecules by using a cone-and-plate shearing device or high-velocity flow through microchannels. Using a syringe pump, cell suspensions were flowed through microchannels of 50 -300 μm diameter drilled through Mylar® sheets using an excimer laser. As quantified by flow cytometry, intracellular uptake and loss of viability correlated with the average shear stress. Optimal results were observed when exposing the cells to high shear stress for short durations in conical channels, which yielded uptake to over one third of cells while maintaining viability at approximately 80%. This method was capable of loading cells with molecules including calcein (0.62 kDa), large molecule weight dextrans (150 -2000 kDa), and bovine serum albumin (66 kDa). These results supported the hypothesis that shearinduced intracellular uptake could be generated by flow of cell suspensions through microchannels and further led to the design of a simple, inexpensive, and effective device to deliver molecules into cells. Such a device could benefit biological research and the biotechnology industry.
Acoustic cavitation has been shown to deliver molecules into viable cells, which is of interest for drug and gene delivery applications. To address mechanisms of these acoustic bioeffects, this work measured the lifetime of albumin-stabilized cavitation bubbles (Optison) and correlated it with desirable (intracellular uptake of molecules) and undesirable (loss of cell viability) bioeffects. Optison was exposed to 500 kHz ultrasound (acoustic pressures of 0.6-3.0 MPa and energy exposures of 0.2-200 J/cm2) either with or without the presence of DU145 prostate cancer cells (10(6) cells/ml) bathed in calcein, a cell-impermeant tracer molecule. Bubble lifetime was determined using a Coulter counter and flow cytometer, while bioeffects were evaluated by flow cytometry. The lifetime of Optison cavitation nuclei was found to decrease and bioeffects (molecular uptake and loss of cell viability) were found to increase with increasing acoustic energy exposure. These bioeffects correlated well with the disappearance of bubbles, suggesting that contrast agent destruction either directly or indirectly affected cells, probably involving unstabilized cavitation nuclei created upon the destruction of Optison. Because Optison solutions presonicated to destroy all detectable bubbles also caused significant bioeffects, the indirect mechanism involving secondary cavitation bubbles is more likely.
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