Active Ingredient: Doxycycline
The mobile phase consisted of water with 0. The encapsulation efficiency of the particles was determined as described previously and found to be approx.
Drug treatments of B. RPMI-1640 medium was prepared such that the final concentrations of each drug in nanoparticles were 195, 49. Control medium contained 0. Individual worms that were previously placed into 48 well flat bottom culture plates upon arrival had new fresh media that contained 1 mL of RPMI-1640.
The nanoparticle-encapsulated drugs and standard antifilarials i. Controls received equal amounts of media but lacked the nanoparticles and standard antifilarials.
Viability assessment Reversibility assays where conducted 24 h following a 9 day course of treatment and or after the final individual within the soluble group reached a motility score of 0.
After 24 h a motility score was recorded.
The same female worms used in the motility assay were gently blotted and transferred to a 96 well microtiter plate that contained 0.
The formazan formed was extracted in 0. The mean absorbance of the formazan from the treated worms was compared with that of the controls. The viability of the treated worms was assessed by calculating the percent inhibition in motility and MTT reduction over the DMSO-treated control worms.
MF shedding and motility The effect of treatments on MF shedding and subsequent motility was assessed using two experimental approaches.
The direct effect on MF was performed by collecting MF from the spent media of healthy untreated female worms. Spent media was centrifuged for 10 min at 200 rcf, the MF were re-suspended in fresh media and enumerated by counting using a 14.
Aliquots containing at least 250 MF were dispensed into wells of a 96 well plate and treated in triplicate with either soluble drug, blank nanoparticles or nanoparticles containing drugs as indicated. Wells were then monitored and scored daily for MF movement.
Motility of MF scoring was carried out using a 10 X Nikon microscope and individual scores from five separate fields of view FOV were averaged to yield a motility score for each well.
Scoring was adapted from a previously reported method by observing the movements of the anterior and posterior body over one min.
In separate experiments, the effect of treating adult female Brugia and their ability to release MF was monitored and recorded for 14 days. The motility of the shed MF was also recorded.
Each experiment, the various treatment groups, and the different doses of the drugs were repeated for a minimum of three times.
Nanoparticle uptake by B. Worms were removed from treatment wells, washed twice in pH 7.
Laser power and exposure settings were set according to the negative worms with no rhodamine and positive rhodamine containing nanoparticles and maintained constant over all the experiments.
Image stacks were collected simultaneously for bright field transmitted light—grey scale, cell nuclei DAPI—blue and nanoparticles rhodamine—red.
Bright field and cell nuclei were used to identify various tissues, organs and structures in the worm such as oral opening, pharynx, uterus, the nerve ring, extracellular secretory apparatus, esophageal track and anal pore.