Megakaryocyte-membrane wrapped nanoparticles for targeted delivery to hematopoietic stem cells
Date
2018
Authors
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Publisher
University of Delaware
Abstract
Hematopoietic Stem and Progenitor Cells (HSPCs) are promising targets for gene therapy as they are self-renewing and differentiate into many blood-related cells. However, they are considered very difficult to transfect. Current delivery methods, such as viral vectors (i.e. lentiviruses), produce poor gene delivery as well as cytotoxicity. However, synthetic PLGA Nanoparticles (NPs) produce low toxicity and can encapsulate genetic cargo. To improve targeted delivery to HSCs in vivo, PLGA NPs can be decorated with the cell membrane removed from Megakaryocytes (Mks) or Mk-derived Microparticles (MkMPs), which we have previously shown to interact with HSCs in vitro and in vivo (Escobar 2017, Jiang 2017). A NP disguised with a Mk membrane will be able to avoid innate immune responses and specifically target HSPCs in vivo where it will deliver its genetic cargo once taken in by the HSPC. ☐ Here, we demonstrate that the protocol for extracting the membranes from Mks to generate Mk-membrane vesicles (MkMVs) has been optimized, that PLGA NPs are successfully wrapped to produce Mk-membrane wrapped NPs (MkNPs), and we explore the mechanism by which MkNPs enter the cell. Wrapping is characterized by Transmission Electron Microscope and internalization of MkNPs in the cytoplasm of HSPCs is seen after 24hrs of introduction in vitro. ☐ In this study, novel methods such as Nanoparticle Tracking Analysis (NTA) and flow cytometry have been used to further characterized MkNPs; these methods have not been utilized by other published literature. Size and concentration of the MkNPs was determined by NTA. As wrapped NPs are too small for detection on flow cytometry, magnetic beads capturing MkNPs were used to confirm wrapping. ☐ The methods developed and optimized in this study will be used in the future to generate Mk-wrapped cargo-loaded NPs for targeted delivery to HSPCs.