@article{e0768738278844f08c4dc7c09a7dbc65,
title = "In vitro and in vivo metabolic tagging and modulation of platelets",
abstract = "Platelets play a critical role in hemostasis at sites of injury and are capable of interacting with various types of cells in the bloodstream. The promise of utilizing platelets for diagnostic and therapeutic applications has motivated the development of facile strategies to functionalize platelets. However, platelets with a small size, lack of nucleus and efficient protein machinery, and low tolerance to chemicals and transfection agents have posed significant challenges for chemical or genetic engineering. Here, for the first time, we report successful metabolic glycan labeling of platelets to introduce chemical tags (e.g., azido groups) onto the membrane of platelets. We demonstrate that azido-sugars can metabolically label platelets in a concentration dependent manner, with cell-surface azido groups detectable at as early as 4 hours. The cell-surface azido groups enable the conjugation of various macromolecular cargos including proteins and polymers onto platelets via efficient click chemistry. Small-molecule drugs such as doxorubicin can also be conjugated onto azido-labeled platelets and become subsequently released to kill surrounding cancer cells, demonstrating the feasibility of utilizing platelets as a drug delivery vehicle. We further show that azido-sugars, upon intraperitoneal injection, can metabolically label platelets with azido groups in vivo, which persist for up to 4 days in mice (nearly the life-span of murine platelets). This in vitro and in vivo platelet labeling and targeting technology opens a new avenue to platelet-based diagnostics and therapeutics.",
keywords = "Cell engineering, Click chemistry, Metabolic glycan labeling, platelet, Sugar",
author = "Dhyanesh Baskaran and Yusheng Liu and Jiadiao Zhou and Yueji Wang and Daniel Nguyen and Hua Wang",
note = "We next studied the stability of cell-surface azido groups on platelets. Platelets were incubated with 100 \u03BCM Ac4ManAz or PBS for 24 h, and then transferred to Ac4ManAz-free fresh media. At 6 h post the media change, Ac4ManAz-treated platelets still exhibited a decent azido density on the cell surface (Fig. 2a\u2013b). At 12 h, Cy5 fluorescence intensity of Ac4ManAz- and PBS-treated platelets after DBCO-Cy5 staining showed negligible differences (Fig. 2a\u2013b), indicating the decay of azido density on the surface of platelets. It is noteworthy that the treatment of platelets with Ac4ManAz did not induce any decrease in platelet viability compared to PBS treatment (Fig. 2c). This is supported by the mitochondria potential assay, which showed negligible differences in the vitality of mitochondria between Ac4ManAz and PBS groups (Fig. S5a). We also incubated platelets with Ac4ManAz or PBS for 24 h, 48 h, and 72 h, respectively, and stained them with Annexin-APC and Calcein AM to detect the necrotic population (Annexin+Calcein AM\u2212). As a result, negligible differences in the fraction of necrotic cells were detected between Ac4ManAz and PBS groups (Fig. S5b). To study whether Ac4ManAz treatment has any effect on the activation status of platelets, we analyzed the surface expression of CD62p, an activation marker of platelets, via flow cytometry. At 12 h, Ac4ManAz treatment did not induce any noticeable change on CD62p expression levels of platelets in comparison with PBS treatment (Fig. 2d). At 24 or 48 h, Ac4ManAz-treated platelets showed reduced CD62p expression in comparison with PBS-treated platelets, indicating the lower activation status of platelets at later times (Fig. 2d). We also performed a clotting assay using collagen-coated glass slides. 2.5 mM Ca2+ was added to the platelet media and then platelets were added onto collagen-coated or non-coated glass slides. As a result, platelets pretreated with PBS or Ac4ManAz for 24 h both formed aggregates in 10 min on the collagen-coated glass slide (Fig. S6). These data demonstrated that 24-h treatment with Ac4ManAz does not induce the necrosis of platelets or impair the coagulation capabilities and vitality of platelets.The authors would like to acknowledge the financial support from NSF DMR 2143673 CAR, NIH R01CA274738, NIH R21CA270872, and the start-up package from the Department of Materials Science and Engineering at the University of Illinois at Urbana-Champaign and the Cancer Center at Illinois. Research reported in this publication was supported by the Cancer Scholars for Translational and Applied Research (C\u2217STAR) Program sponsored by the Cancer Center at Illinois and the Carle Cancer Center under Award Number CST EP012023.",
year = "2025",
month = jun,
doi = "10.1016/j.mtbio.2025.101719",
language = "English (US)",
volume = "32",
journal = "Materials Today Bio",
issn = "2590-0064",
publisher = "Elsevier B.V.",
}