DBS Assay with LC-MS/MS for the Determination of Idelalisib, A Selective PI3K-δ Inhibitor in Mice Blood and Its Application to a Pharmacokinetic Study.

Idelalisib is a selective and second-generation PI3K-δ inhibitor, approved for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this paper, we present a fully validated dried blood spot (DBS) method for the quantitation of idelalisib from mice blood using an LC-MS/MS, which was operated under multiple reaction monitoring mode. To the punched DBS discs, acidified methanol enriched with internal standard (IS; larotrectinib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of idelalisib and the IS was achieved on an Atlantis dC18 column using a mixture of 10 mM ammonium formate:acetonitrile (25:75, v/v). The flow-rate and injection volume were 0.80 mL/min and 2.0 µL, respectively. Idelalisib and the IS were eluted at ~0.98 and 0.93 min, respectively and the total run time was 2.00 min. Idelalisib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 416.1→176.1 and 429.1→342.1, respectively was used for the quantitation. The calibration range was 1.01-4 797 ng/mL. No matrix effect and carry over were observed. Haematocrit did not influence DBS idelalisib concentrations. All the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mice pharmacokinetic study.

A dried blood spot assay with HPLC-MS/MS for the determination of larotrectinib in mouse blood and its application to a pharmacokinetic study.

Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC-MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC18 column using 10 mm ammonium formate-acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transitions of 429.1 → 342.1 and 474.1 → 267.1, respectively, were used for the quantitation. The calibration range was 1.06-5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study.

Mapping the Coulomb Environment in Interference-Quenched Ballistic Nanowires.

The conductance of semiconductor nanowires is strongly dependent on their electrostatic history because of the overwhelming influence of charged surface and interface states on electron confinement and scattering. We show that InAs nanowire field-effect transistor devices can be conditioned to suppress resonances that obscure quantized conduction thereby revealing as many as six sub-bands in the conductance spectra as the Fermi-level is swept across the sub-band energies. The energy level spectra extracted from conductance, coupled with detailed modeling shows the significance of the interface state charge distribution revealing the Coulomb landscape of the nanowire device. Inclusion of self-consistent Coulomb potentials, the measured geometrical shape of the nanowire, the gate geometry and nonparabolicity of the conduction band provide a quantitative and accurate description of the confinement potential and resulting energy level structure. Surfaces of the nanowire terminated by HfO2 are shown to have their interface donor density reduced by a factor of 30 signifying the passivating role played by HfO2.

Photocatalytic Applications of Electrospun TiO2 Nanofibres Embedded with Bimodal Sized and Prismatic Gold Nanoparticles.

In this paper, we have synthesized electrospun TiO2 nanofibers embedded with bimodal sized and prismatic gold nanoparticles. The surface plasmons generated in the gold nanoparticles were used to enhance the performance of photocatalysis. The photocatalytic conversion efficiencies of these bimodal sized/prismatic gold nanoparticles when embedded in electrospun TiO2 fibres showed an enhancement of upto 60% over bare fiber systems and also show higher efficiencies than electrospun fibrous systems embedded with unimodal sized gold nanoparticles. Anisotropic bimodal gold nanoparticles show the highest degree of photocatalytic activity. This may be attributed to greater density/concentration of nanoparticles with higher effective surface area and formation of a junction between the smaller and larger nanoparticles. Such a bimodally distributed range of nanoparticles could also lead to greater trapping of charge carriers at the TiO2 conduction band edge and promoting catalytic reactions on account of these trapped charges. This enhanced photocatalytic activity is explained by invoking different operating mechanisms such as improved surface area, greater trapping, coarse plasmon resonance and band effects. Thus, a useful applicability of the gold nanoparticles is shown in the area of photocatalysis.

Fabrication of poly (L-lactic acid)/gelatin composite tubular scaffolds for vascular tissue engineering.

The in vitro fabrication of fully functional 3D vascular tissue construct represents one of the most fundamental challenges in vascular tissue engineering. Polymer blending is an effective method for developing, desirable bio-composites for tissue engineering. This study employs the blending of desired characteristics of a synthetic polymer, poly (L-lactic acid) (PLLA) and a biopolymer, gelatin for enhancing cell adhesion sites. Aligned and random PLLA/gelatin nanofibers were fabricated using electrospinning technique. Morphological and chemical characterization of the nanofibrous scaffolds was carried out and the size of fibers ranged from 100 to 500 nm. The SEM, fluorescent staining and viability assays revealed an increase in viability and proliferation of Human Umbilical Vein Endothelial Cells (HUVECs) and Smooth Muscle Cells (SMCs) proportional to gelatin content. The aligned fiber morphology helps cells to orient and elongate along their long axis. Thus the results were suggestive of the fact that topographically aligned nanofibrous scaffolds control cellular organization and possibly provide a good support for achieving the vital organization and physical properties of blood vessel.

Determination of Trace Metals, Moisture, pH and Assessment of Potential Toxicity of Selected Smokeless Tobacco Products.

The characterization and classification of smokeless tobacco products has been a continuously evolving process. This is based on a number of different parameters like nicotine content, moisture content, amount of heavy metals, pH, and in vitro cytotoxicity assays. Their contexts often vary between countries, research institutions, and legal requirements. The categorisation of these products is quite challenging due to the diffused sample sizes, diverse array of branded products on offer, and the absence of a centralized manufacturing facility. This study aims at a systematic classification of 10 smokeless tobacco product samples from the retail market based on their potential toxicity upon long-term use. The estimation of potential toxicity follows a well-established method that employs the concentration of toxic metals in the different samples. The potential toxicity as well as heavy metal concentrations of the smokeless tobacco products analysed was found to be much higher than acceptable limits. For instance, the levels of lead, cadmium, copper and zinc of 2.5, 1, 4 and 23 ppm, respectively, are well above their recommended limits. The results from the study indicate that chronic use of smokeless tobacco products is a significant health risk, especially in the vulnerable population. Further studies of this nature will help establish a toxicological fingerprint on the diverse class of products that floods the market now.

Fabrication of fibrin based electrospun multiscale composite scaffold for tissue engineering applications.

Fabricating scaffolds mimicking the native extracellular matrix (ECM) in both structure and function is a key challenge in the field of tissue engineering. Previously we have demonstrated a novel electrospinnig method for the fabrication of fibrin nanofibers using Poly(vinyl alcohol) (PVA) as an 'electrospinning-driving' polymer. Here we demonstrate the fabrication and characterization of a multiscale fibrin based composite scaffold with polycaprolactone (PCL) by sequential electrospinning of PCL microfibers and fibrin nanofibers. This multiscale scaffold has great potential for tissue engineering applications due to the combined benefits of biological nanofibers such as cell attachment and proliferation and that of microfibers such as open structure, larger pore size and adequate mechanical strength. Physico chemical characterization of the electrospun scaffold was done by Scanning Electron Microscopy (SEM), Contact angle analysis, fibrin specific Phosphotungstic acid haematoxyllin (PTAH) staining and evaluation of mechanical properties. SEM data revealed the formation of bead free nanofibers of fibrin with a fiber diameter ranging from 50-500 nm and microfibers of PCL in the size range of 1 microns to 2.5 microns. These dimensions mimic the hierarchical structure of ECM found in native tissues. Cell attachment and viability studies using human mesenchymal stem cells (hMSC) revealed that the scaffold is non toxic and supports cell attachment, spreading and proliferation. In addition, we examined the inflammatory potential of the scaffold to demonstrate its usefulness in tissue engineering applications.

Gelatin nanoparticles loaded poly(ε-caprolactone) nanofibrous semi-synthetic scaffolds for bone tissue engineering.

Nanofibrous semi-synthetic polymeric nanocomposite scaffolds were engineered by incorporating a maximum of 15 wt% biopolymeric gelatin nanoparticles (nGs) into the synthetic polymer poly(ε-caprolactone) (PCL) prior to electrospinning. The effect of nGs in altering the physico-chemical properties, cell material interaction and biodegradability of the scaffolds was evaluated. Experimental results showed that the inherent hydrophobicity of PCL scaffolds remained unaltered even after the incorporation of hydrophilic nGs. However, breakdown of the continuous nanofibers into lengths less than 7 µm occurred within four to eight weeks in the presence of nGs in contrast with the greater than two year time frame for the degradation of PCL fibers alone that is known from the literature. In terms of cell-material interaction, human mesenchymal stem cells (hMSCs) were found to attach and spread better and faster on PCL_nG scaffolds compared to PCL scaffolds. However, there was no difference in hMSC proliferation and differentiation into osteogenic lineage between the scaffolds. These results indicate that PCL_nG nanofibrous nanocomposite scaffolds are an improvement over PCL scaffolds for bone tissue engineering applications in that the PCL_nG scaffolds provide improved cell interaction and are able to degrade and resorb more efficiently.

Fabrication of aligned poly(lactic acid)-chitosan nanofibers by novel parallel blade collector method for skin tissue engineering.

Poly(lactic acid) (PLA) was blended with chitosan (CS) to fabricate electrospun aligned PLA-CS nanofibers. These prepared nanofibers were aligned using a novel collector made of parallel blades which is designed to increase the transversal electric field across the gap. SEM images show that the fiber diameter mostly ranges between 150 +/- 60 nm and Fourier Transform infrared Spectroscopy (FTIR) analysis confirm the presence of PLA and CS. X-Ray Diffraction (XRD) studies explains the amorphous nature of electrospun PLA-CS nanofibers, suitable for faster degradation. Degradation studies confirmed that PLA-CS nanofiber has enhanced degradation than the pure PLA fibers. Cell studies with human dermal fibroblasts (HDF) show the orientation of cells along the direction of fiber alignment. The results indicate that the prepared PLA-CS aligned nanofibers are promising material for skin tissue engineering.

5-flourouracil loaded N,O-carboxymethyl chitosan nanoparticles as an anticancer nanomedicine for breast cancer.

Chitosan and its carboxymethyl derivatives are smart biopolymers that are non-toxic, biocompatible, biodegradable and hence found applications in biomedical field. In the current work, we have developed 5-fluorouracil (5-FU) loaded N,Ocarboxymethyl chitosan (N,O-CMC) nanoparticles (mean diameter: 80 +/- 20 nm, zeta potential: +52.47 +/- 2 mV) for cancer drug delivery. Drug entrapment efficiency (65%) and in vitro drug release studies were carried out spectrophotometricaly. Cellular internalization of the drug loaded nanoparticles was confirmed by fluorescent microscopy and flow cytometric analysis. Results of anticancer activity via MTT, apoptosis and caspase 3 assays showed the toxicity of the drug loaded nanoparticles towards breast cancer cells. As a whole these results indicates the potential of 5-FU loaded N,O-CMC nanoparticles in breast cancer chemotherapy in which the side effects of conventional chemo treatment could be reduced. Furthermore, the results of in vitro hemolytic assay and coagulation assay substantiate the blood compatibility of the system as well.