Cell membrane disintegration and extracellular vesicle release in a model of different size and charge PAMAM dendrimers effects on cultured endothelial cells

Abstract

Different nanomaterials are under development for various biomedical applications in which nanoparticles contact blood and vasculature. Therefore, investigating the interactions between nanomaterials and vascular endothelial cells (ECs) is of great importance. Here, we show the effects of polyamidoamine (PAMAM) dendrimers of two different sizes, generation 2 (G2; approximately 3 nm diameter) and generation 7 (G7; 9 nm), with neutral (OH-terminated), anionic (COOH-terminated), and cationic (NH₂-terminated) surface modifications on cultured human umbilical vein ECs (HUVECs). We found that only cationic dendrimers (5–100 μg/mL G7-NH₂ and 100 µg/mL G2-NH₂) and not anionic or neutral dendrimers were cytotoxic to HUVECs. In addition, cationic dendrimers at low concentrations (5 μg/mL) markedly increased the HUVEC surface expression of the proinflammatory activation marker ICAM-1 and phosphatidylserine (PS). Both G2-NH₂ and G7-NH₂ dendrimers caused g1 arrest, but only G7-NH₂ dendrimers induced significant HUVEC apoptosis. G7-NH₂ interacted strongly with HUVEC plasma membranes and mitochondrial membranes, and phospholipid vesicles containing G7-NH₂ formed, which resulted in extensive plasma membrane blebbing and disintegration. Furthermore, flow cytometric analysis showed that G7-NH₂-treated HUVECs released large numbers of extracellular vesicles (EVs) positive for CD105 and PS. A notable population of EVs positive for the mitochondrial marker TOM20 but negative for the autophagosome marker LC3 was found. In summary, large cationic PAMAM dendrimers (G7-NH₂) showed both proinflammatory and proapoptotic effects in ECs; at high dendrimer concentrations, these effects were accompanied by necrotic cytotoxicity. G7-NH₂ caused plasma and mitochondrial membrane disintegration and the release of EVs, including EVs of mitochondrial origin that were not associated with mitophagy.

Keywords:

• Nanoparticle toxicity
• apoptosis
• inflammation
• mitochondria
• flow cytometry
• extracellular vesicles

Acknowledgments

This project was supported in part by appointments to the Research Participation Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

Disclaimer

The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The study was funded by an FDA Office of Chief Scientist Collaborative Opportunities for Research Excellence in Science (CORES) internal grant and by the internal research funds of the Center for Biologics Evaluation and Research, FDA.