Vaccines, Blood & Biologics

Investigation of Potential Toxic Effects of Engineered Nanoparticles and Biologic Microparticles in Blood and Their Biomarker Applications.

Principal Investigator: Jan Simak, PhD
Office / Division / Lab: OBRR / DBCD / LCH

General Overview

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. One important class of nanomaterials is made of carbon atoms. Because of their unique and useful properties, carbon nanomaterials are under investigation for various applications in biomedical nanotechnologies, usually as structures called carbon fullerenes and carbon nanotubes (CNTs).

The superior mechanical characteristics of CNTs make them very attractive materials for making devices for collecting, processing, and storing blood transfusion products, diagnostic biosensors (devices that use biological material such as enzymes, cells, or antibodies to detect a substance), drug delivery systems, and imaging devices for use inside blood vessels. Several studies, however, are raising safety concerns by showing toxic effects of fullerenes and CNTs. Therefore, one part of our research is focused on determining the mechanism of toxicity in these studies, by evaluating whether these nanomaterials are toxic to blood vessels and blood cells.

Our investigation is focused specifically on the effects of various commercially available carbon nanomaterials on the outer membranes of blood cells and platelets that we isolate from blood and also on vessel wall cells grown in tissue cultures. This research will help FDA to evaluate the safety and effectiveness of different nanomaterials for medical use.

We are also studying cell membrane microparticles (MPs) in blood. MPs are microscopic vesicles (membrane-enclosed sacs) released from the outer membrane of blood cells, platelets, and blood vessel cells. MPs are present in the circulating blood of healthy donors; rises in the levels of MPs in the blood are associated with diseases that damage blood vessels.

Moreover, because MPs are released from blood cells during processing and storage of cellular blood components, the potential for MPs to cause adverse events following the administration of blood products is also a concern. Among the major potential adverse events are abnormal clotting and inflammation, particularly after administration of blood platelet products. In fact, moderate to severe reactions to transfusion of platelet products have been documented in more than 20% of recipients.

In addition, there is need for new quality control tests to detect MPs in stored and treated cellular blood products and to evaluate blood transfusion devices. New MP tests for blood could also significantly contribute to the protection of public health by improving the diagnosis of injury or stimulation of blood vessels, blood cells, and platelets (stimulation of platelets triggers clotting). This will apply to blood and cardiovascular diseases, infections, and other diseases and conditions that injure blood vessels, blood cells, or platelets.

Scientific Overview

The first part of our research program is focused on investigation of blood and vascular biocompatibility of carbon nanomaterials.

Carbon nanomaterials, particularly carbon fullerenes and nanotubes (CNTs), have a unique combination of properties that make them among the most useful materials for biomedical nanotechnologies. Their superior mechanical characteristics make them very attractive candidates for constructing devices for collecting, processing, and storing blood transfusion products. Moreover, CNTs are critical elements of diagnostic biosensors, drug delivery nanosystems, and imaging nanoprobes for intravascular use. However, the rapid development of biomedical nanotechnology poses serious challenges to the safety evaluation of novel nanoscale materials; several studies have raised safety concerns by showing cytotoxic effects of fullerenes and CNTs. Thus the investigation of effects of fullerenes and CNTs on blood cells, platelets and endothelial cells is a critical safety issue. The results of our Critical Path research project on blood and vascular biocompatibility of carbon nanomaterials will help FDA to create regulatory policy for evaluating the safety and effectiveness of different biomedical nanomaterials.

Our investigation is focused on effects of various commercially available carbon nanomaterials and their structural and chemical derivatives on plasma membranes of cultured endothelial cells, blood cells, and platelets isolated from blood. We use a variety of cell biology assays to study in vitro cytotoxicity of these materials, such as apoptosis, necrosis, autophagy, effects on the cell cycle, cell surface activation markers, and intracellular calcium concentration.

The second part of this program is focused on investigating cell membrane microparticles (MPs) in blood. MPs in blood are small vesicles of 0.1-1.0 μm released from the plasma membrane of different cell types, particularly from platelets, blood cells and endothelial cells. MPs are present in circulating blood of healthy donors and their elevated counts are associated with various disease states. Moreover, there is concern that MPs released from blood cells during processing and storage of cellular blood components pose potential causes of prothrombotic and proinflammatory adverse events following blood product administration. In fact, moderate to severe reactions to transfusion have been documented in more than 20% of individuals receiving platelet products.

Development and validation of methods for MP analysis are essential for quality control of stored and treated cellular blood products, and also for evaluating blood transfusion devices. New assays for cell membrane microparticle analysis in blood could also significantly contribute to protecting public health by assisting in the diagnosis of injury or stimulation of endothelial cells, blood cells, or platelets. This will apply to hematologic and cardiovascular diseases, infections, and other pathologies with a vascular injury component. We use a variety of immunodetection methods in these studies, including flow cytometry and light and electron microscopy.


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

J Biol Chem 2014 Oct 17;289(42):29247-60
First demonstration of transmissible spongiform encephalopathy-associated prion protein (PrPTSE) in extracellular vesicles from plasma of mice infected with mouse-adapted variant Creutzfeldt-Jakob disease by in vitro amplification.
Saa P, Yakovleva O, de Castro J, Vasilyeva I, De Paoli SH, Simak J, Cervenakova L

Biomaterials 2014 Aug;35(24):6182-94
The effect of protein corona composition on the interaction of carbon nanotubes with human blood platelets.
De Paoli SH, Diduch LL, Tegegn TZ, Orecna M, Strader MB, Karnaukhova E, Bonevich JE, Holada K, Simak J

Nanomedicine 2014 Jul;10(5):939-48
Toxicity of carboxylated carbon nanotubes in endothelial cells is attenuated by stimulation of the autophagic flux with the release of nanomaterial in autophagic vesicles.
Orecna M, De Paoli SH, Janouskova O, Tegegn TZ, Filipova M, Bonevich JE, Holada K, Simak J

Biomed Opt Express 2012 Jun 1;3(6):1312-25
Multimodal optical studies of single and clustered colloidal quantum dots for the long-term optical property evaluation of quantum dot-based molecular imaging phantoms.
Kang H, Clarke ML, Lacerda SH, Karim A, Pease LF 3rd, Hwang J

Mol Pharm 2012 Mar 5;9(3):382-93
Nanoparticle Size and Surface Charge Determine Effects of PAMAM Dendrimers on Human Platelets in Vitro.
Dobrovolskaia MA, Patri AK, Simak J, Hall JB, Semberova J, De Paoli Lacerda SH, McNeil SE

ACS Nano 2011 Jul 26;5(7):5808-13
Carbon nanotubes activate store-operated calcium entry in human blood platelets.
De Paoli Lacerda SH, Semberova J, Holada K, Simakova O, Hudson SD, Simak J

Transfusion 2011 May;51(5):1012-21
Underestimation of the expression of cellular prion protein on human red blood cells.
Panigaj M, Brouckova A, Glierova H, Dvorakova E, Simak J, Vostal JG, Holada K

Biologicals 2010 Sep;38(5):602-11
Meeting report on protein particles and immunogenicity of therapeutic proteins: filling in the gaps in risk evaluation and mitigation.
Carpenter J, Cherney B, Lubinecki A, Ma S, Marszal E, Mire-Sluis A, Nikolai T, Novak J, Ragheb J, Simak J

Langmuir 2010 Jul 6;26(13):11581-8
Microfluidic directed self-assembly of liposome-hydrogel hybrid nanoparticles.
Hong JS, Stavis SM, DePaoli Lacerda SH, Locascio LE, Raghavan SR, Gaitan M

ACS Nano 2010 Jan 26;4(1):365-79
Interaction of Gold Nanoparticles with Common Human Blood Proteins.
Lacerda SH, Park JJ, Meuse C, Pristinski D, Becker ML, Karim A, Douglas JF

Nano Lett 2009 Sep;9(9):3312-7
Carbon nanotubes activate blood platelets by inducing extracellular Ca(2+) influx sensitive to calcium entry inhibitors.
Semberova J, De Paoli Lacerda SH, Simakova O, Holada K, Gelderman MP, Simak J

Methods Mol Biol 2008;484:79-93
Flow cytometric analysis of cell membrane microparticles.
Gelderman MP, Simak J

Int J Nanomedicine 2008;3(1):59-68
Adverse effects of fullerenes on endothelial cells: fullerenol C60(OH)24 induced tissue factor and ICAM-I membrane expression and apoptosis in vitro.
Gelderman MP, Simakova O, Clogston JD, Patri AK, Siddiqui SF, Cvostal A, Simak J

Ir J Med Sci 2008 Mar;177(1):11-7
Critically ill newborns with multiple organ dysfunction: assessment by NEOMOD score in a tertiary NICU.
Janota J, Simak J, Stranak Z, Matthews T, Clarke T, Corcoran D

Arterioscler Thromb Vasc Biol 2007 Jul;27(7):e138-9
Elevated endothelial microparticles in Fabry children decreased after enzyme replacement therapy.
Gelderman MP, Schiffmann R, Simak J

Haematologica 2006 Aug;91(8):1126-9
Expression of cellular prion protein on platelets from patients with gray platelet or Hermansky-Pudlak syndrome and the protein's association with alpha-granules.
Holada K, Glierova H, Simak J, Vostal JG

J Thromb Haemost 2006 Jun;4(6):1296-302
Circulating endothelial microparticles in acute ischemic stroke: a link to severity, lesion volume and outcome.
Simak J, Gelderman MP, Yu H, Wright V, Baird AE

Transfus Med Rev 2006 Jan;20(1):1-26
Cell membrane microparticles in blood and blood products: potentially pathogenic agents and diagnostic markers.
Simak J, Gelderman MP

Circulation 2005 Jan 18;111(2):212-21
Using peripheral blood mononuclear cells to determine a gene expression profile of acute ischemic stroke: a pilot investigation.
Moore DF, Li H, Jeffries N, Wright V, Cooper RA Jr, Elkahloun A, Gelderman MP, Zudaire E, Blevins G, Yu H, Goldin E, Baird AE



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