Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions.

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Coating and Pharmacokinetic Evaluation of Air Spray Coated Drug Coated Balloons.

Drug coated balloons (DCB) are becoming the standard-care treatment for peripheral arterial disease (PAD). DCB use excipients to transfer and retain anti-proliferative drugs, such as paclitaxel. Excipients thus play a vital role in the design and function of DCB, however methods to coat balloons with excipients and anti-proliferative drugs remain unknown. The goal of this study was to thus develop an approach to coat and evaluate DCB for various excipients. An air sprayer method was developed to deposit paclitaxel and various excipients onto non-coated commercially available angioplasty balloons. The coating of the angioplasty balloons was evaluated for drug deposition and coating efficiency using high performance liquid chromatography tandem mass spectrometry. Drug transfer and retention of the coated angioplasty balloons into arterial segments were evaluated ex vivo using harvested pig arteries in a pulsatile flow bioreactor. The air sprayer method successfully delivered varying excipients including bovine serum albumin (BSA), urea and iohexol. The air spray method was configured to coat four angioplasty balloons simultaneously with paclitaxel and iohexol with an average paclitaxel load of 4.0 ± 0.70 µg/mm2. The intra-day (within) and inter-day (between) coating precisions, defined as relative standard deviation (RSD), was 17.2 and 15.5%, respectively. Ex vivo deployment of iohexol-paclitaxel DCB yielded an arterial paclitaxel concentration of 123.4 ± 44.68 ng/mg (n = 3) at 1 h, 126.7 ± 25.27 ng/mg (n = 3) at 1 day, and 12.9 ± 12.88 ng/mg (n = 3) at 7 days. This work provides proof-of-concept of a quick, inexpensive approach to coat commercially available angioplasty balloons with paclitaxel and various excipients.

HPLC-MS/MS method for quantification of paclitaxel from keratin containing samples.

Local drug delivery of paclitaxel is becoming ever more prevalent. As complex drug/excipient combinations are being developed and tested, new high performance liquid chromatography-mass spectrometry (HPLC-MS) techniques capable of quantifying paclitaxel from such formulations are needed. Here a method for quantifying paclitaxel from aqueous, protein and oil containing samples was developed and validated. Keratin, derived from human hair, is the protein component/paclitaxel excipient in the development and validation of said method. The novelty of this method is described by its ability to overcome water solubility issues and address clean-up of residual solvents in clinical grade paclitaxel injection composition. The method evaluates tert-butyl methyl ether and ethanol as extraction solvents with an extraction efficiency of 31.9±2.3% and 86.4±4.5% respectively. Upon evaporation and rehydration, samples were evaluated by HPLC-MS and a method was developed for paclitaxel quantification. The method developed had an inter-day precision of 9.1% relative standard deviation and an intra-day precision of 4.3% relative standard deviation normalized to a docetaxel internal standard. The described method is applicable to any aqueous paclitaxel sample containing protein and/or oils.