Probing ADP Induced Aggregation Kinetics During Platelet-Nanoparticle Interactions: Functional Dynamics Analysis to Rationalize Safety and Benefits.

Platelets, one of the most sensitive blood cells, can be activated by a range of external and internal stimuli including physical, chemical, physiological, and/or non-physiological agents. Platelets need to respond promptly during injury to maintain blood hemostasis. The time profile of platelet aggregation is very complex, especially in the presence of the agonist adenosine 5'-diphosphate (ADP), and it is difficult to probe such complexity using traditional linear dose response models. In the present study, we explored functional analysis techniques to characterize the pattern of platelet aggregation over time in response to nanoparticle induced perturbations. This has obviated the need to represent the pattern of aggregation by a single summary measure and allowed us to treat the entire aggregation profile over time, as the response. The modeling was performed in a flexible manner, without any imposition of shape restrictions on the curve, allowing smooth platelet aggregation over time. The use of a probabilistic framework not only allowed statistical prediction and inference of the aggregation signatures, but also provided a novel method for the estimation of higher order derivatives of the curve, thereby allowing plausible estimation of the extent and rate of platelet aggregation kinetics over time. In the present study, we focused on the estimated first derivative of the curve, obtained from the platelet optical aggregometric profile over time and used it to discern the underlying kinetics as well as to study the effects of ADP dosage and perturbation with gold nanoparticles. In addition, our method allowed the quantification of the extent of inter-individual signature variations. Our findings indicated several hidden features and showed a mixture of zero and first order kinetics interrupted by a metastable zero order ADP dose dependent process. In addition, we showed that the two first order kinetic constants were ADP dependent. However, we were able to perturb the overall kinetic pattern using gold nanoparticles, which resulted in autocatalytic aggregation with a higher aggregate mass and which facilitated the aggregation rate.

Intracellular iron overload leading to DNA damage of lymphocytes and immune dysfunction in thalassemia major patients.

OBJECTIVES: To investigate the cause and effects of intracellular iron overload in lymphocytes of thalassemia major patients. METHODS: Sixty-six thalassemia major patients having iron overload and 10 age-matched controls were chosen for the study. Blood sample was collected, and serum ferritin, oxidative stress; lymphocyte DNA damage were examined, and infective episodes were also counted. RESULTS: Case-control analysis revealed significant oxidative stress, iron overload, DNA damage, and rate of infections in thalassemia cases as compared to controls. For cases, oxidative stress (ROS) and iron overload (serum ferritin) showed good correlation with R2  = 0.934 and correlation between DNA damage and ROS gave R2  = 0.961. We also demonstrated that intracellular iron overload in thalassemia caused oxidative damage of lymphocyte DNA as exhibited by DNA damage assay. The inference is further confirmed by partial inhibition of such damage by chelation of iron and the concurrent lowering of the ROS level in the presence of chelator deferasirox. CONCLUSION: Therefore, intracellular iron overload caused DNA fragmentation, which may ultimately hamper lymphocyte function, and this may contribute to immune dysfunction and increased susceptibility to infections in thalassemia patients as indicated by the good correlation (R2  = 0.91) between lymphocyte DNA damage and rate of infection found in this study.

Mahanine, a DNA minor groove binding agent exerts cellular cytotoxicity with involvement of C-7-OH and -NH functional groups.

Mahanine, a carbazole alkaloid is a potent anticancer molecule. To recognize the structure-activity correlation, mahanine was chemically modified. Antiproliferative activity of these derivatives was determined in 19 cancer cell lines from 7 different origins. Mahanine showed enhanced apoptosis compared to dehydroxy-mahanine-treated cells, indicating significant contribution of the C-7-OH group. O-Methylated-mahanine and N-methylated dehydroxy-mahanine-treated cells exhibited apoptosis only at higher concentrations, suggesting additional contribution of 9-NH group. Using biophysical techniques, we demonstrated that mahanine interacts with DNA through strong association with phosphate backbone compared to other derivatives but is unable to induce any conformational change in DNA, hence suggesting the possibility of being a minor groove binder. This was corroborated by molecular modeling and isothermal titration calorimetry studies. Taken together, the results of the current study represent the first evidence of involvement of C-7-OH and 9-NH group of mahanine for its cytotoxicity and its minor groove binding ability with DNA.

Superparamagnetic iron oxide nanoparticle attachment on array of micro test tubes and microbeakers formed on p-type silicon substrate for biosensor applications.

A uniformly distributed array of micro test tubes and microbeakers is formed on a p-type silicon substrate with tunable cross-section and distance of separation by anodic etching of the silicon wafer in N, N-dimethylformamide and hydrofluoric acid, which essentially leads to the formation of macroporous silicon templates. A reasonable control over the dimensions of the structures could be achieved by tailoring the formation parameters, primarily the wafer resistivity. For a micro test tube, the cross-section (i.e., the pore size) as well as the distance of separation between two adjacent test tubes (i.e., inter-pore distance) is typically approximately 1 µm, whereas, for a microbeaker the pore size exceeds 1.5 µm and the inter-pore distance could be less than 100 nm. We successfully synthesized superparamagnetic iron oxide nanoparticles (SPIONs), with average particle size approximately 20 nm and attached them on the porous silicon chip surface as well as on the pore walls. Such SPION-coated arrays of micro test tubes and microbeakers are potential candidates for biosensors because of the biocompatibility of both silicon and SPIONs. As acquisition of data via microarray is an essential attribute of high throughput bio-sensing, the proposed nanostructured array may be a promising step in this direction.