Investigation of Potential Toxic Effects of Engineered Nanoparticles and Biologic Microparticles in Blood and Their Biomarker Applications
Jan Simak, Ph.D.
Office of Blood Research and Review
Division of Blood Components and Devices
Laboratory of Cellular Hematology
Dr. Simak is a Principal Investigator at the Laboratory of Cellular Hematology, Division of Blood Components and Devices, Office of Blood Research and Review, CBER. Dr. Simak received his Ph.D. in Biochemistry in 1992 at the Institute of Hematology and Blood Transfusion / University of Chemistry and Technology in Prague, Czech Republic. In 1992 – 2000, he served as Assistant Professor and Principal Investigator at the Department of Pathophysiology, 1st Faculty of Medicine of the Charles University (Prague), and at the Department of Neonatal Intensive Care, Institute for the Care of Mother and Child (Prague). Dr. Simak joined the Laboratory of Cellular Hematology at CBER in February 2000. In 2002, he established a CBER research program to investigate extracellular membrane vesicles in blood and blood products. This work expanded to include several collaborative projects on the toxic effects of engineered nanomaterials on blood platelets and vascular endothelial cells (supported by the FDA Office of Chief Scientist Collaborative Opportunities for Research Excellence in Science [CORES] grants). Most recently, Dr. Simak’s group has used its expertise in nanoscale analysis and characterization of blood platelet membranes to study platelet cryopreservation. In addition to pursuing regulatory science research, Dr. Simak is a product reviewer of regulatory submissions related to cellular blood products and transfusion devices. Moreover, Dr. Simak is a CBER representative in the FDA Nanotechnology Taskforce.
Blood transfusion products, including red blood cell, platelet, and plasma products, contain microscopic phospholipid vesicles (membrane enclosed sacs). These are also called membrane microparticles or extracellular membrane vesicles (EVs), and they are released in blood from membranes of blood cells, platelets, and blood vessel cells. Blood cells and platelets also release membrane microparticles during processing and storage of cellular blood components used to treat people. The potential for EVs to cause adverse events following the administration of blood products is a concern. Among the major potential adverse events are abnormal clotting and stimulation of inflammation, particularly after administration of blood platelet products. Moreover, platelet products manufactured for a long storage by freezing or by freeze-drying contain large amounts of membrane microparticles, which stimulate blood clotting. Their potential role in associated adverse events has not been elucidated yet. Therefore, the first part of our research program is focused on characterization of membrane microparticles in blood and blood products, as well as studies of their biological activities. Since most of the membrane particles of interest are too small for analysis by conventional methods, we are also working on development of high resolution analytical and imaging assays to characterize membrane vesicles and other lipid and protein particles at nano scale.
Nanoscience analytical techniques are also employed in the second part of our research program, which is focused on investigation of blood and vascular toxicity of engineered nanomaterials. Nanotechnology is the science that enables scientists to create, explore, and manipulate materials that are measured in nanometers (billionths of a meter), that is, tens of thousands of times smaller than the width of a human hair. Such objects, called nanomaterials, can have chemical, physical, and biological properties that differ from those of their larger counterparts of the same chemical composition. Materials of various compositions, including metals, carbon, silica, synthetic polymers, lipids, or proteins, may be engineered at nanoscale to create nanoparticles of different sizes, shapes, or surfaces with various nanoscale patterns. Nanomaterials have characteristics that make them attractive for various biomedical applications. Several studies, however, raise safety concerns by detecting toxic effects of different nanomaterials in blood. Therefore, our research is focused on determining the mechanisms of toxicity, such as by evaluating which characteristics make nanomaterials toxic to blood cells and blood vessels. Our investigation is specifically focused on the effects of various nanomaterials on platelets and blood cells that we isolate from blood. We are also studying blood proteins involved in blood clotting and vessel wall cells grown in tissue culture. This research helps FDA scientists evaluate the safety and effectiveness of different nanomaterials being applied for medical use. In addition, our laboratory is also conducting collaborative studies on models for evaluation of toxicity of subvisible particles naturally occurring in blood products and plasma derivatives by using engineered nanoparticles coated with different proteins or lipids. Finally, we are also exploring applications of engineered nanoparticles as cryoprotectants for platelet cold storage and cryopreservation.
Most biologics, including blood products, contain wide-spectrum submicron phospholipid membrane particles and aggregated protein particles, which may affect product safety and efficacy. A leading project of our research program is focused on application and development of high-resolution nanoscience analytical and imaging methods for analysis of membrane micro- and nanovesicles in blood products and evaluation of their biological activities in vitro. Cell membrane extracellular vesicles (EVs) are microscopic vesicles, 40 – 1000 nm in size, released in blood from membranes of blood cells, platelets, vascular endothelial cells and other cell types. Different populations of EVs are present in the circulating blood of healthy donors; rises in the levels of EVs in the blood are associated with various diseases.
Since various populations of EVs are present in cellular blood components and their counts increase during blood product processing and storage, it is important to elucidate how these EVs affect efficacy and safety of blood products. We are particularly interested in whether they contribute to transfusion-associated adverse events. Among the major potential adverse events are prothrombotic and proinflammatory effects, particularly after administration of blood platelet products. In fact, moderate to severe reactions to transfusion of platelet products have been documented in over 20% of recipients. In addition, in frozen and freeze-dried platelet-derived hemostatic products, submicron EVs represent a major component with a marked impact on the product procoagulant potency in vitro and potentially also on safety and efficacy in vivo. More than 90% of EVs present in blood and blood products are smaller than 300 nm, most commonly of exosome size (40 – 100 nm). These nanoscale EVs are not detectable by traditional analytical methods, such as conventional flow cytometry. Thus, there is an urgent need for reliable quality control methods to analyze membrane micro- and nanovesicles in blood products.
The second part of our research program is focused on blood and vascular toxicity studies of engineered nanoparticles. Different types of engineered nanoparticles are employed in developing drug delivery systems or vaccines. Further, nanomaterials are useful in diagnostic biosensors or theranostic devices, including imaging devices for use inside blood vessels. Engineered nanomaterials are likely to eventually find wide applications in devices for collecting, processing, and storing blood transfusion products. Several studies, however, raise safety concerns about blood and vascular toxicity of nanomaterials. Therefore, our investigations are specifically focused on the effects of various nanomaterials on platelets and cultured endothelial cells. We are also engaged in collaborative studies on characterization and biological effects of nanoparticle protein corona formed by different plasma proteins. Engineered nanoparticles coated with different proteins or lipids serve as very useful models to investigate toxic effects of protein and lipid particles that occur naturally in biologic products. Finally, our latest project is focused on investigation of certain engineered nanoparticles as cryoprotectants for platelet cold storage and cryopreservation.
- ORCID ID: 0000-0001-8910-7362
- Innovation and Regulatory Science- Research Summary: FDA scientists discover key chemical steps in formation of microparticles carrying abnormal hemoglobin in sickle cell disease
- Innovation and Regulatory Science- Scientific Poster: Microparticles from sickle cell disease RBCs
- Nanotoxicology 2019 Jun;13(5):664-81
Cell membrane disintegration and extracellular vesicle release in a model of different size and charge PAMAM dendrimers effects on cultured endothelial cells.
Patel M, De Paoli SH, Elhelu OK, Farooq S, Simak J
- JCI Insight 2018 Nov 2;3(21):e120451
Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles.
Jana S, Strader MB, Meng F, Hicks W, Kassa T, Tarandovskiy I, De Paoli S, Simak J, Heaven MR, Belcher JD, Vercellotti GM, Alayash AI
- Cell Mol Life Sci 2018 Oct;75(20):3781-801
Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome.
De Paoli SH, Tegegn TZ, Elhelu OK, Strader MB, Patel M, Diduch LL, Tarandovskiy ID, Wu Y, Zheng J, Ovanesov MV, Alayash A, Simak J
- PLoS One 2018 Oct 31;13(10):e0206557
An effective "three-in-one" screening assay for testing drug and nanoparticle toxicity in human endothelial cells.
Filipova M, Elhelu OK, De Paoli SH, Fremuntova Z, Mosko T, Cmarko D, Simak J, Holada K
- Wiley Interdiscip Rev Nanomed Nanobiotechnol 2017 Sep;9(5):e1448
The effects of nanomaterials on blood coagulation in hemostasis and thrombosis.
Simak J, De Paoli S
- J Extracellular Vesicles 2016 May 4;5:30422
Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets.
Tegegn TZ, De Paoli SH, Orecna M, Elhelu OK, Woodle SA, Tarandovskiy ID, Ovanesov MV, Simak J
- J Pharm Sci 2016 Mar;105(3):1023-7
Subvisible particle content, formulation, and dose of an erythropoietin peptide mimetic product are associated with severe adverse postmarketing events.
Kotarek J, Stuart C, De Paoli SH, Simak J, Lin TL, Gao Y, Ovanesov M, Liang Y, Scott D, Brown J, Bai Y, Metcalfe DD, Marszal E, Ragheb JA
- Transfusion 2015 Nov;55(11):2590-6
Expression of the cellular prion protein affects posttransfusion recovery and survival of red blood cells in mice.
Glier H, Simak J, Panigaj M, Gelderman MP, Vostal JG, Holada K