The goal of this proposal is to design new approaches and bench-top devices that enable high throughput separation and purification of exosomes from minimally processed patient serum samples.
The aim i s to produce highly purified materials that are devoid of genetic, proteomic, or lipidomic moieties exogenous to exosomal structures. Exosomes are natural biocompatible nanovesicles whose in vivo function is poorly understood given the difficulty of isolating large quantities of uniformly disperse exosome subpopulations for experimentation. In spite of this hurdle, new efforts have begun to focus on the high potential utility of exosomes as therapeutic nano-carriers. In contrast to artificial nanovesicles, cell-derived exosomes express a plethora of complex targeting ligands including integrins, receptors or even tetraspanins in native conformations and contain a cytoskeleton allowing for deformability. Consequently, exosomes are showing promise as cancer vaccines and have even been modified to deliver siRNA across the blood brain barrier. For these reasons, investigating means to study exosome function in vivo for the purposes of understanding their role in disease pathogenesis or adapt exosomes for use as therapeutic delivery vehicles is warranted. We are proposing to develop an instrument for separation and purification of exosomes without any labels such that these nanovesicles can be collected, modified and used as drug carriers without affecting their inherent biocompatibility and functionality. This work will employ field-flow fractionation (FFF) and split-flow lateral thin transport (SPLITT) techniques to characterize and continuously separate exosomes from cell cultures and identified serum and/or plasma samples. PI Gale, with expertise in FFF and microfluidics, has teamed up with co-PIs Wickline and Hood who are experts in exosome biology, to develop an instrument capable of characterizing exosomes based on biophysical properties and continuously separate exosomes without any labels or any structural/functional damage. The following are the specific aims for this project:
Specific Aim 1 : To characterize exosomes and identify optimal separation conditions using FFF.
Specific Aim 2 : To fabricate and test SPLITT and serial SPLITT systems for continuous exosome separation.
Specific Aim 3 : To demonstrate continuous separation of cultured and serum and/or plasma exosomes with serial SPLITT system.
Exosomes are natural biocompatible nanovesicles whose in vivo function is poorly understood given the difficulty with isolating large quantities of uniforml disperse exosome subpopulations for experimentation. We propose to develop field-flow fractionation based instruments for the separation and characterization of exosomes as therapeutic agents. We will apply electrical, flow, and sedimentation FFF techniques, along with appropriate microfluidic technologies to separate exosomes from whole blood. We will work with exosome biology experts at Washington University in St. Louis to complete this work.
|Bardi, Gina T; Smith, Mary Ann; Hood, Joshua L (2018) Melanoma exosomes promote mixed M1 and M2 macrophage polarization. Cytokine 105:63-72|
|Ornthai, Mathuros; Siripinyanond, Atitaya; Gale, Bruce K (2016) Biased cyclical electrical field-flow fractionation for separation of submicron particles. Anal Bioanal Chem 408:855-63|
|Hood, Joshua L (2016) Post isolation modification of exosomes for nanomedicine applications. Nanomedicine (Lond) 11:1745-56|
|Ornthai, Mathuros; Siripinyanond, Atitaya; Gale, Bruce K (2016) Effect of Ionic and Nonionic Carriers in Electrical Field-Flow Fractionation. Anal Chem 88:1794-803|
|Hood, Joshua L (2016) Melanoma exosomes enable tumor tolerance in lymph nodes. Med Hypotheses 90:11-3|
|Hood, Joshua L (2016) Melanoma exosome induction of endothelial cell GM-CSF in pre-metastatic lymph nodes may result in different M1 and M2 macrophage mediated angiogenic processes. Med Hypotheses 94:118-22|
|Hu, Lingzhi; Wickline, Samuel A; Hood, Joshua L (2015) Magnetic resonance imaging of melanoma exosomes in lymph nodes. Magn Reson Med 74:266-271|
|Petersen, Kevin E; Manangon, Eliana; Hood, Joshua L et al. (2014) A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM. Anal Bioanal Chem 406:7855-66|