Radiation synthesized protein-based nanoparticles: A technique overview Gustavo H.C. Varca a,n , Gabriela G. Perossi a , Mariano Grasselli b , Ademar B. Lugão a a Instituto de Pesquisas Energéticas e Nucleares, Centro de Quimica e Meio Ambiente (IPEN-CNEN/SP), Av. Prof. Lineu Prestes, no. 2242, Cidade Universitária, Zip Code, 05508-000 São Paulo, SP, Brazil b Universidad Nacional de Quilmes– IMBICE (CONICET), Roque Sáenz Peña 352, Bernal, B1876BXD Buenos Aires, Argentina HIGHLIGHTS  Synthesis of protein-based nanoparticles by γ-irradiation.  Optimization of the technique.  Overview of mechanism involved in the nanoparticle formation.  Engineered papain nanoparticles for biomedical applications. article info Article history: Received 31 January 2014 Accepted 11 May 2014 Available online 23 May 2014 Keywords: Protein-based nanoparticle Papain Ionizing radiation Protease Protein-crosslinking abstract Seeking for alternative routes for protein engineering a novel technique – radiation induced synthesis of protein nanoparticles – to achieve size controlled particles with preserved bioactivity has been recently reported. This work aimed to evaluate different process conditions to optimize and provide an overview of the technique using γ-irradiation. Papain was used as model protease and the samples were irradiated in a gamma cell irradiator in phosphate buffer (pH¼7.0) containing ethanol (0–35%). The dose effect was evaluated by exposure to distinct γ-irradiation doses (2.5, 5, 7.5 and 10 kGy) and scale up experiments involving distinct protein concentrations (12.5–50 mg mL 1 ) were also performed. Characterization involved size monitoring using dynamic light scattering. Bityrosine detection was performed using fluorescence measurements in order to provide experimental evidence of the mechanism involved. Best dose effects were achieved at 10 kGy with regard to size and no relevant changes were observed as a function of papain concentration, highlighting very broad operational concentration range. Bityrosine changes were identified for the samples as a function of the process confirming that such linkages play an important role in the nanoparticle formation. & 2014 Elsevier Ltd. All rights reserved. 1. Introduction Proteins as a whole represent an important group of thera- peutic agents available nowadays for the treatment of a wide range of disorders. Despite direct applications, these biomolecules may also be used to functionalize, confer biopharmaceutical advantages and constitute novel drug delivery systems for avail- able drugs among other aspects (Banta et al., 2010; Sezaki and Hashida, 1985). Perhaps the most relevant aspect to be taken into account with regard to globular proteins in pharmaceutics and industrial pro- cesses is attributed to instability in unusual environments and intrinsic limitations of such biomolecules. Thus, many approaches have been directed towards overcoming such limitations (Polizzi et al., 2007) including nanotechnological tools (Crommelin et al., 2003), chemical modifications (Fernandez-Lafuente et al., 1995), immobilization (Sheldon, 2007), use of additives such as sugars (Arakawa and Timasheff, 1982; Varca et al., 2010) among others, considering that overcoming such problems and intrinsic limita- tions would allow a great expansion in the use of such compounds (Arnold, 1993). Particularly the use of high energy radiation is known to directly or indirectly damage or impair biological function of macromolecules and proteins (Saha et al., 1995; Davies, 1987; Furuta, 2002) and as a result its use is therefore limited. However, over the last decade some researchers have attempted to use radiation (Akiyama et al., 2007; Furusawa et al., 2004) to achieve nanometer-sized particles and nanogels based on proteins and Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/radphyschem Radiation Physics and Chemistry http://dx.doi.org/10.1016/j.radphyschem.2014.05.020 0969-806X/& 2014 Elsevier Ltd. All rights reserved. n Corresponding author. Tel.: þ55 11 3133 9250. E-mail address: varca@usp.br (G.H.C. Varca). Radiation Physics and Chemistry 105 (2014) 48–52