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Considerations and Some Practical Solutions to Overcome Nanoparticle Interference with LAL Assays and to Avoid Endotoxin Contamination in Nanoformulations

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Characterization of Nanoparticles Intended for Drug Delivery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1682))

Abstract

Monitoring endotoxin contamination in drugs and medical devices is required to avoid pyrogenic response and septic shock in patients receiving these products. Endotoxin contamination of engineered nanomaterials and nanotechnology-based medical products represents a significant translational hurdle. Nanoparticles often interfere with an in vitro Limulus Amebocyte Lysate (LAL) assay commonly used in the pharmaceutical industry for the detection and quantification of endotoxin. Such interference challenges the preclinical development of nanotechnology-formulated drugs and medical devices containing engineered nanomaterials. Protocols for analysis of nanoparticles using LAL assays have been reported before. Here, we discuss considerations for selecting an LAL format and describe a few experimental approaches for overcoming nanoparticle interference with the LAL assays to obtain more accurate estimation of endotoxin contamination in nanotechnology-based products. The discussed approaches do not solve all types of nanoparticle interference with the LAL assays but could be used as a starting point to address the problem. This chapter also describes approaches to prevent endotoxin contamination in nanotechnology-formulated products.

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References

  1. Brade H, Opal SM, Vogel SN, Morrison DC (eds) (1999) Endotoxin in health and disease. Marcel Dekker Inc., New York

    Google Scholar 

  2. Dobrovolskaia MA, Vogel SN (2002) Toll receptors, CD14, and macrophage activation and deactivation by LPS. Microbes Infect 4(9):903–914. doi:10.1016/S1286-4579(02)01613-1

    Article  CAS  PubMed  Google Scholar 

  3. USP 30 NF 25 (2007) <85> Bacterial endotoxins test. vol 1

    Google Scholar 

  4. HHS, US FDA (2012) Guidance for industry. Pyrogen and endotoxins testing: questions and answers. https://www.fda.gov/downloads/drugs/guidances/ucm310098.pdf

  5. HHS, US FDA (2015) .Guidance for Industry and Food and Drug Administration Staff. Endotoxin testing recommendations for single-use intraocular ophthalmic devices. https://www.fda.gov/downloads/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm393376.pdf

  6. Jones CF, Grainger DW (2009) In vitro assessments of nanomaterial toxicity. Adv Drug Deliv Rev 61(6):438–456. doi:10.1016/j.addr.2009.03.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sharma SK (1986) Endotoxin detection and elimination in biotechnology. Biotechnol Appl Biochem 8(1):5–22

    CAS  PubMed  Google Scholar 

  8. Crist RM, Grossman JH, Patri AK, Stern ST, Dobrovolskaia MA, Adiseshaiah PP, Clogston JD, McNeil SE (2013) Common pitfalls in nanotechnology: lessons learned from NCI’s Nanotechnology Characterization Laboratory. Integr Biol (Camb) 5(1):66–73. doi:10.1039/c2ib20117h

    Article  CAS  Google Scholar 

  9. Dobrovolskaia MA, Patri AK, Potter TM, Rodriguez JC, Hall JB, McNeil SE (2012) Dendrimer-induced leukocyte procoagulant activity depends on particle size and surface charge. Nanomedicine (Lond) 7(2):245–256. doi:10.2217/nnm.11.105

    Article  CAS  Google Scholar 

  10. Inoue K (2011) Promoting effects of nanoparticles/materials on sensitive lung inflammatory diseases. Environ Health Prev Med 16(3):139–143. doi:10.1007/s12199-010-0177-7

    Article  CAS  PubMed  Google Scholar 

  11. Inoue K, Takano H (2011) Aggravating impact of nanoparticles on immune-mediated pulmonary inflammation. Scientific World J 11:382–390. doi:10.1100/tsw.2011.44

    Article  CAS  Google Scholar 

  12. Inoue K, Takano H, Yanagisawa R, Hirano S, Kobayashi T, Fujitani Y, Shimada A, Yoshikawa T (2007) Effects of inhaled nanoparticles on acute lung injury induced by lipopolysaccharide in mice. Toxicology 238(2–3):99–110. doi:10.1016/j.tox.2007.05.022

    Article  CAS  PubMed  Google Scholar 

  13. Inoue K, Takano H, Yanagisawa R, Hirano S, Sakurai M, Shimada A, Yoshikawa T (2006) Effects of airway exposure to nanoparticles on lung inflammation induced by bacterial endotoxin in mice. Environ Health Perspect 114(9):1325–1330. doi:10.1289/ehp.8903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Shi Y, Yadav S, Wang F, Wang H (2010) Endotoxin promotes adverse effects of amorphous silica nanoparticles on lung epithelial cells in vitro. J Toxicol Environ Health A 73(11):748–756. doi:10.1080/15287391003614042

    Article  CAS  PubMed  Google Scholar 

  15. Dobrovolskaia MA, McNeil SE (2016) Nanoparticles and endotoxin. In: Dobrovolskaia MA, McNeil SE (eds) Handbook of immunological properties of engineered nanomaterials, vol 1. World Scientific Publishing, Singapore, pp 143–187

    Chapter  Google Scholar 

  16. Alwis KU, Milton DK (2006) Recombinant factor C assay for measuring endotoxin in house dust: comparison with LAL, and (1 --> 3)-beta-D-glucans. Am J Ind Med 49(4):296–300. doi:10.1002/ajim.20264

    Article  CAS  PubMed  Google Scholar 

  17. Ding JL, Ho B (2010) Endotoxin detection--from limulus amebocyte lysate to recombinant factor C. Subcell Biochem 53:187–208. doi:10.1007/978-90-481-9078-2_9

    Article  CAS  PubMed  Google Scholar 

  18. McKenzie JH, Alwis KU, Sordillo JE, Kalluri KS, Milton DK (2011) Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay. J Environ Monit 13(6):1739–1745. doi:10.1039/c1em10035a

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Fujita Y, Nabetani T (2014) Iron sulfate inhibits Limulus activity by induction of structural and qualitative changes in lipid A. J Appl Microbiol 116(1):89–99. doi:10.1111/jam.12349

    Article  CAS  PubMed  Google Scholar 

  20. Reich J, Lang P, Grallert H, Motschmann H (2016) Masking of endotoxin in surfactant samples: effects on Limulus-based detection systems. Biologicals 44(5):417–422. doi:10.1016/j.biologicals.2016.04.012

    Article  CAS  PubMed  Google Scholar 

  21. Lyons JL, Roos KL, Marr KA, Neumann H, Trivedi JB, Kimbrough DJ, Steiner L, Thakur KT, Harrison DM, Zhang SX (2013) Cerebrospinal fluid (1,3)-beta-D-glucan detection as an aid for diagnosis of iatrogenic fungal meningitis. J Clin Microbiol 51(4):1285–1287. doi:10.1128/jcm.00061-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tran T, Beal SG (2016) Application of the 1,3-beta-D-Glucan (Fungitell) assay in the diagnosis of invasive fungal infections. Arch Pathol Lab Med 140(2):181–185. doi:10.5858/arpa.2014-0230-RS

    Article  PubMed  Google Scholar 

  23. Henne W, Schulze H, Pelger M, Tretzel J, von Sengbusch G (1984) Hollow-fiber dialyzers and their pyrogenicity testing by Limulus amebocyte lysate. Artif Organs 8(3):299–305

    Article  CAS  PubMed  Google Scholar 

  24. Neun BW, Dobrovolskaia MA (2011) Detection and quantitative evaluation of endotoxin contamination in nanoparticle formulations by LAL-based assays. Methods Mol Biol 697:121–130. doi:10.1007/978-1-60327-198-1_12

    Article  CAS  PubMed  Google Scholar 

  25. Sandle T (2011) A practical approach to depyrogenation studies using bacterial endotoxin. J GxP Compliance 15(4):90–96

    Google Scholar 

  26. Subbarao N (2016) Nanoparticle sterility and sterilization of nanomaterials. In: Dobrovolskaia MA, McNeil SE (eds) Handbook of immunological properties of engineered nanomaterials, vol 1 and 6. World Scientific Publishing Ltd, Singapore, pp 53–75

    Chapter  Google Scholar 

  27. Zheng J, Clogston JD, Patri AK, Dobrovolskaia MA, McNeil SE (2011) Sterilization of silver nanoparticles using standard gamma irradiation procedure affects particle integrity and biocompatibility. J Nanomed Nanotechnol 2011(Suppl 5):001. doi:10.4172/2157-7439.s5-001

    PubMed  PubMed Central  Google Scholar 

  28. Ragab AA, Van De Motter R, Lavish SA, Goldberg VM, Ninomiya JT, Carlin CR, Greenfield EM (1999) Measurement and removal of adherent endotoxin from titanium particles and implant surfaces. J Orthop Res 17(6):803–809. doi:10.1002/jor.1100170603

    Article  CAS  PubMed  Google Scholar 

  29. Dobrovolskaia MA, Neun BW, Clogston JD, Ding H, Ljubimova J, McNeil SE (2010) Ambiguities in applying traditional Limulus amebocyte lysate tests to quantify endotoxin in nanoparticle formulations. Nanomedicine (Lond) 5(4):555–562. doi:10.2217/nnm.10.29

    Article  CAS  Google Scholar 

  30. London AS, Mackay K, Lihon M, He Y, Alabi BR (2014) Gel filtration chromatography as a method for removing bacterial endotoxin from antibody preparations. Biotechnol Prog 30(6):1497–1501. doi:10.1002/btpr.1961

    Article  CAS  PubMed  Google Scholar 

  31. Ma R, Zhao J, Du HC, Tian S, Li LW (2012) Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction. Anal Biochem 424(2):124–126. doi:10.1016/j.ab.2012.02.015

    Article  CAS  PubMed  Google Scholar 

  32. Mack L, Brill B, Delis N, Groner B (2014) Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags. Anal Biochem 466:83–88. doi:10.1016/j.ab.2014.08.020

    Article  CAS  PubMed  Google Scholar 

  33. Magalhaes PO, Lopes AM, Mazzola PG, Rangel-Yagui C, Penna TC, Pessoa A Jr (2007) Methods of endotoxin removal from biological preparations: a review. J Pharm Pharm Sci 10(3):388–404

    PubMed  Google Scholar 

  34. Afonin KA, Grabow WW, Walker FM, Bindewald E, Dobrovolskaia MA, Shapiro BA, Jaeger L (2011) Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine. Nat Protoc 6(12):2022–2034. doi:10.1038/nprot.2011.418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Dobrovolskaia MA, Germolec DR, Weaver JL (2009) Evaluation of nanoparticle immunotoxicity. Nat Nanotechnol 4(7):411–414. doi:10.1038/nnano.2009.175

    Article  CAS  PubMed  Google Scholar 

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Acknowledgment

This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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Authors and Affiliations

  1. Cancer Research Technology Program¸ Nanotechnology Characterization Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, P.O. Box B, Frederick, MD, 21702, USA

    Barry W. Neun & Marina A. Dobrovolskaia

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  1. Barry W. Neun
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  2. Marina A. Dobrovolskaia
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Corresponding author

Correspondence to Marina A. Dobrovolskaia .

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Editors and Affiliations

  1. Nanotechnology Characterization Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

    Scott E. McNeil

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Neun, B.W., Dobrovolskaia, M.A. (2018). Considerations and Some Practical Solutions to Overcome Nanoparticle Interference with LAL Assays and to Avoid Endotoxin Contamination in Nanoformulations. In: McNeil, S. (eds) Characterization of Nanoparticles Intended for Drug Delivery. Methods in Molecular Biology, vol 1682. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7352-1_3

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  • DOI: https://doi.org/10.1007/978-1-4939-7352-1_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7350-7

  • Online ISBN: 978-1-4939-7352-1

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