Assessing the potential of lipid nanoparticles in the capture of polychlorinated biphenyls (PCBs)

Methods

Summary

This project will follow a similar basic format of the methods outlined in the following preprint; "Investigation of DPPC liposomes reveals their capability to entrap Aroclor 1260, an emerging environmental pollutant".

Not only will this study expand on the results reported in this manuscript, it will serve to verify and promote reproducibility in the methods described.  

Challenges

Anticipated challenges that I foresee, and have experienced, are managing nanoparticle stability. Nanoparticles begin to show signs of deterioration after approximately 24 hours at room temperature, which was previously observed while establishing the current protocol for benchtop handling of LNP samples (see preprint above). To eliminate issues of stability, I intend to prepare LNPs fresh for each analysis and/or store them at 4 degC not longer than 8-10 hours. The initial chosen concentration range for HCBP is based on previous experimentation, and for this analysis HCBP quantities may exceed 1.0 mg.  Importantly, quantities far exceeding 1 mg per sample may no longer realistically reflect what would be found in the environment. I also plan to incorporate microscopy (if necessary) to assess sample integrity for longer periods of storage, although, this may necessitate additional time and funding. LNPs prepared with DPPC can range in size from 400 to 520 nm depending on the preparation conditions and other factors described in the following paper [ref]. 

Pre Analysis Plan

Lipid nanoparticles can be readily captured using centrifugal concentrators with a molecular weight cutoff below the size of the lipid nanoparticles. Incorporated HCBP can be extracted by adding a small volume of ethanol to the recovered LNPs, which will disrupt the nanoparticles and release any captured HCBP. Further, ethanol will solublize the HCBP (which is minimally water soluble) enabling spectroscopic measurements to be collected. The lipids do not absorb appreciably at a wavelength of 236 nm, where HCBP strongly absorbs. A standard curve of HCBP will be prepared in ethanol to enable quantitation from our samples. Appropriate sample controls (buffer w/ HCBP but no LNPs, and buffer w/o HCBP but w/ LNPs) are outlined in the preprint referenced above and will be used here as well.