Blood and Vascular Biocompatibility of Biologic and Engineered Micro/Nanoparticles: The Role of Cell Membrane Microparticles in Adverse Events and as Biomarkers

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

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Overview

Public Health Issue: Rapid development of nanotechnology brings serious challenges on safety evaluation of novel nanoscale materials. Due to a unique combination of their physico-chemical properties, carbon nanomaterials, particularly carbon fullerenes and nanotubes (CNTs) are among the principal materials used in biomedical nanotechnologies. First part of this program is focused on investigation of blood and vascular biocompatibility of carbon nanomaterials. Superior mechanical characteristics like elasticity and tensile strength make CNTs very attractive candidates as materials for plastic composites for various biomedical applications including devices for collection, processing, and storage of blood transfusion products. Moreover, CNTs have a profound impact on the development of diagnostic biosensors, drug delivery nanosystems, or imaging nanoprobes for intravascular use. Several studies, however, are raising 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. Second part of this program is focused on investigation of cell membrane microparticles (MPs) in blood. MPs of 0.1-1.0 [micro]m in size derived from platelets, blood cells and endothelial cells are present in circulating blood of healthy donors and their elevated counts are associated with various disease states. Moreover, the potential role of MP in adverse events following blood product administration is also of concern, since MPs are released from blood cells and platelets during processing and storage of cellular blood components. Major adverse event concerns are prothrombotic (clotting) and proinflammatory effects, particularly after administration of platelet products where moderate to severe reactions to transfusion have been documented in more than 20% of recipients.

Regulatory Contribution: The FDA Nanotechnology Task Force Report (July 2007)recommends strengthening FDA's promotion of, and participation in,research and other efforts to increase scientific understanding to facilitate assessment of data needs for regulated products. This would include promoting efforts, and participating in collaborative efforts, to further understand biological interactions of nanoscale materials, including, as appropriate the development of data to assess likelihood of long term health effects from exposure to specific nanoscale materials; Assessing data on general particle interactions with biological systems and on specific particles of concern to FDA. The investigation of blood and vascular biocompatibility of carbon nanoparticles is a Critical Path project contributing to meet these FDA goals. Similarly, investigation of cell membrane microparticles in blood and blood products has a significant regulatory impact. The FDA recommends quantitation of MP as a supplemental test for in vitro characterization of platelet products (Guidance for Industry For Platelet Testing and Evaluation of Platelet Substitute Products, CBER FDA 1999). However, there is no standard method for MP analysis available. The results on MP analysis in different laboratories differ signifificantly. Finally, since elevated MP in circulation may reflect blood cell activation and/or vascular injury, circulating MP may become valuable biomarkers for in vivo evaluation of biologics, drugs, and devices.Besides monitoring of blood cell stimulation and vascular injury in various diseases, MP analysis may be used as a QC marker for in vitro testing of blood products. Therefore, development and validation of immunodetection and functional assays for MP analysis in blood is essential. Screening of MP phenotypes for diagnostic value is needed.

Research Approach: The first part of our program is focused on investigation of in vitro effects of carbon nanomaterials on plasma membranes of cultured endothelial cells and blood cells and platelets isolated from blood. Various commercially available carbon nanomaterials, specifically carbon fullerenes and nanotubes, and their structural and chemical derivatives are studied. Different cell biology assays are used to study in vitro cytotoxicity - apoptosis, necrosis, autophagy; effects on cell cycle, cell surface activation markers, intracellular calcium concentration etc. The second part of the program is focused on the development of assays for analysis of MP in plasma using a three-color flow cytometric method. Using this method, we have demonstrated elevated counts of endothelial MP in patients with different vascular pathologies such as paroxysmal nocturnal hemoglobinuria, sickle cell disease, Fabry disease, and acute stroke. We also use this method to analyze MP in blood products, e.g., in platelet units collected by the apheresis method in blood centers nationwide. Our preliminary results showed elevated counts of platelet, blood cell, and endothelial cell derived MP, and also MP of procoagulant types in some apheresis platelet units, compared to healthy donor plasma. We will pursue further studies to identify MP phenotypes with potentially adverse vascular effects and investigate the impact of collection, processing, and storage methods on MP levels in apheresis platelets.

Mission Relevance & Outcomes: The results of the Critical Path research project on blood and vascular biocompatibility of carbon nanomaterials will serve the Agency to create regulatory policy for evaluation of safety and effectiveness of different carbon nanotubes and fullerenes for medical use.
Development and validation of assays for cell membrane microparticle analysis in blood could significantly contribute to the protection of public health in the diagnosis of endothelial cell, blood cell and platelet stimulation or injury. This will apply to a variety of diseases such as: hematologic -, infectious-, or cardiovascular diseases and other pathologies with a vascular injury component. MP analysis will be used for investigation of adverse effects of biologics, particularly cellular blood products, frozen or treated with pathogen inactivation techniques. Thus, development and validation of methods for MP analysis are essential not only for quality control of stored and treated cellular blood products, but also for evaluation of blood transfusion devices.

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Publications

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.

Br J Haematol 2004 Jun;125(6):804-13
Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria.
Simak J, Holada K, Risitano AM, Zivny JH, Young NS, Vostal JG.