Browsing by Author "Kao, Chen-Yuan"
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Item Engineering megakaryocytic microparticles for nucleic acid delivery and their biological effects to hematopoietic stem/progenitor cells(University of Delaware, 2019) Kao, Chen-YuanMegakaryocytes (Mks) are large polyploid cells derived from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and give rise to platelets and megakaryocytic microparticles (MkMPs), the most abundant MPs in circulation. MkMPs can induce the differentiation of hematopoietic stem and progenitor cells (HSPCs) into functional Mks. In this study, we show that MkMPs target HSPCs with high specificity. Using fluorescent confocal microscopy and electron microscopy, we identify two mechanisms by which MkMPs deliver cargo to HSPCs: endocytosis, and specifically macropinocytosis and lipid raft-dependent endocytosis, and direct fusion of MkMPs into HSPCs. We show that HSPC uropods are the preferential site for MkMP binding, and that CD54 (ICAM-1), CD11b, CD18 and CD43, localized on HSPC uropods, are involved in MkMP binding to HSPCs. We also investigate the role of miRNAs from MkMP in MkMP-induced Mk differentiation of HSPCs. Our study identified the importance of three miRNAs, miR-486-5p, miR-92a-3p, and miR-22-3p, alone and in combination, in mediating Mk differentiation of HSPCs in the absence of thrombopoietin. Signaling pathways JNK, p38, and PI3K/mTOR are also identified as mediating the MkMP-induced Mk differentiation of HSPCs. ☐ Since HSPCs are important target cells for gene therapy applications, we developed a nonviral system based on MkMPs for targeted delivery of plasmid DNA (pDNA) and small RNAs to HSPCs. With an optimized electroporation protocol, an average of 4200 plasmid copies per MP were loaded into MPs, thus enabling effective delivery of green fluorescent protein (GFP)–encoding pDNA to HSPCs and HSPC nuclei, with up to 81% of the nuclei containing pDNA. Effective functional small interfering RNA (siRNA) and microRNA (miRNA) delivery were also demonstrated. ☐ Finally, we examine the role of p53 in the Mk shear-stress response. We demonstrated that shear flow stimulates p53 acetylation and Caspase 9 activation, and demonstrated that shear-stimulated Caspase 9 activation and Mk particle biogenesis depend on transcription-independent p53-induced apoptosis (TIPA), but PS externalization is not.Item Megakaryocyte membrane-wrapped nanoparticles for targeted cargo delivery to hematopoietic stem and progenitor cells(Bioengineering and Translational Medicine, 2022-11-29) Das, Samik; Harris, Jenna C.; Winter, Erica J.; Kao, Chen-Yuan; Day, Emily S.; Papoutsakis, Eleftherios TerryHematopoietic stem and progenitor cells (HSPCs) are desirable targets for gene therapy but are notoriously difficult to target and transfect. Existing viral vector-based delivery methods are not effective in HSPCs due to their cytotoxicity, limited HSPC uptake and lack of target specificity (tropism). Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are attractive, nontoxic carriers that can encapsulate various cargo and enable its controlled release. To engineer PLGA NP tropism for HSPCs, megakaryocyte (Mk) membranes, which possess HSPC-targeting moieties, were extracted and wrapped around PLGA NPs, producing MkNPs. In vitro, fluorophore-labeled MkNPs are internalized by HSPCs within 24 h and were selectively taken up by HSPCs versus other physiologically related cell types. Using membranes from megakaryoblastic CHRF-288 cells containing the same HSPC-targeting moieties as Mks, CHRF-wrapped NPs (CHNPs) loaded with small interfering RNA facilitated efficient RNA interference upon delivery to HSPCs in vitro. HSPC targeting was conserved in vivo, as poly(ethylene glycol)–PLGA NPs wrapped in CHRF membranes specifically targeted and were taken up by murine bone marrow HSPCs following intravenous administration. These findings suggest that MkNPs and CHNPs are effective and promising vehicles for targeted cargo delivery to HSPCs.