Tuesday, April 18, 2017

mPEG-PLA from PolySciTech used by Yale University in development of a novel blood-circulation assay method

A fundamental difficulty with medicinal applications to humans is that the circulatory systems of most living organisms are designed specifically to screen out any perceived toxins or ‘non-self’ components. Typically, the kidneys and the liver work together along with macrophages (white blood cells) to remove any chemicals or particulates from the bloodstream. Although this system provides protection to the human body from toxic ingestion, it creates great difficulty for applying medicines as it greatly reduces the blood circulation time of medicinal molecules. For general medicinal applications, the loss of drug from the bloodstream is calculated as the circulation half-life and dosing schedules are calculated to match. One method to improve blood-circulation is to encapsulate the drug molecule inside of PEG-PLA so-called ‘stealth’ nanoparticles. For these particles, the PEG external coating prevents attacks by macrophages while the size alone reduces uptake and clearance by kidneys or liver. These particles enhance the blood circulation time of medicines, but a key question is by exactly how much is the blood circulation time enhanced and what is the new circulation half-life. This question critical for practical applications as it would define the dosing schedule of the encapsulated drug as it must be dosed often enough to maintain effect but not too often so as to potentially have toxic side-effects. Recently, researchers at Yale University utilized mPEG-PLA from PolySciTech (www.polyscitech.com) (PolyVivo Cat# AK054) to generate stealth-nanoparticles as test substrates for their novel fluorescence microscopy-based technique for determining half-life of particles using as little as 2 uL of blood. This research holds promise for rapid and routine determination of half-life using very small samples of blood. Read more: Tietjen, Gregory T., Jenna DiRito, Jordan S. Pober, and W. Mark Saltzman. "Quantitative microscopy-based measurements of circulating nanoparticle concentration using microliter blood volumes." Nanomedicine: Nanotechnology, Biology and Medicine (2017). http://www.sciencedirect.com/science/article/pii/S1549963417300643

“Abstract: Nanoparticles (NPs) are potential drug delivery vehicles for treatment of a broad range of diseases. Intravenous (IV) administration, the most common form of delivery, is relatively non-invasive and provides (in theory) access throughout the circulatory system. However, in practice, many IV injected NPs are quickly eliminated by specialized phagocytes in the liver and spleen. Consequently, new materials have been developed with the capacity to significantly extend the circulating half-life of IV administered NPs. Unfortunately, current procedures for measuring circulation half-lives are often expensive, time consuming, and can require large blood volumes that are not compatible with mouse models of disease. Here we describe a simple and reliable procedure for measuring circulation half-life utilizing quantitative microscopy. This method requires only 2 μL of blood and minimal sample preparation, yet provides robust quantitative results. Graphical Abstract: Quantitative microscopy can be used to measure circulating concentrations of nanoparticles with as little as 2 μL of blood. However, when using such small volumes, the path length within and between samples can vary significantly as the high viscosity of blood can yield differences in think layer thickness as the blood spreads following application of a coverslip. This yields variability in the measured mean fluorescence intensity. Addition of a reference nanoparticle of a different color can correct the mean fluorescence intensity variance. Thus, quantitative microscopy can serve as a robust method for measuring nanoparticle half-life using μL volumes of blood. NP formulation: NP were prepared by a standard nanoprecipitation procedure. PLA–PEG (PolyVivo AK054) was dissolved at an initial concentration of 100 mg/mL in DMSO and then diluted to the desired concentration for NP formulation (typically ~55 mg/mL for the ~165 nm NPs used in this study) along with addition of either DiI or DiO dye also dissolved in DMSO. NPs were loaded with DiI or DiO dye at a final wt dye/wt polymer ratio of 0.5%. The dye/polymer solution in DMSO was added drop wise to vigorously stirring sterile diH2O in batches of 200 μL polymer/dye solution added to 1.3 mL of diH2O with identical repetitions performed to generate a full NP batch. NP were subsequently filtered through a 1.2 μm cellulose acetate membrane (GE Healthcare Life Sciences - Whatman) filter to remove any free dye or polymer aggregates and then pooled. Typically, 8 small batches of ~11 mg polymer each were combined for a total pooled batch size of ~88 mg initial polymer weight. The pooled NP solutions were then transferred to a 12 mL volume 10,000 MWCO dialysis cassettes (Thermo Scientific - Slide-A-Lyzer) and dialyzed against 2× exchanges of ~2.2 L of diH2O at room temperature to remove excess DMSO. Following dialysis NPs were aliquoted and snap frozen in liquid N2. One aliquot from each NP batch was lyophilized in a pre-weighed tube in order to determine the NP concentration. Standard NP concentration was typically ~5 mg/mL. NP batches were diluted to ~0.1 mg/mL and analyzed via dynamic light scattering (DLS) to confirm NP size and homogeneity.”


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