Hemoglobin loaded polymeric nanoparticles: Preparation and characterizations Alberto Dessy a,1 , Anna M. Piras a,1 , Giorgio Schirò b , Matteo Levantino b , Antonio Cupane b , Federica Chiellini a,⇑ a Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Application (BIOlab) UdR–INSTM Consortium Reference Center, University of Pisa, Via Livornese 1291, 56122 San Piero a Grado, Pisa, Italy b Department of Physical and Astronomical Sciences, University of Palermo, Via Archirafi 36, I-90123 Palermo, Italy article info Article history: Received 14 January 2011 Received in revised form 8 March 2011 Accepted 23 March 2011 Available online 5 April 2011 Keywords: Polymeric nanoparticles Blood substitutes Biocompatible polymers Injectable systems abstract In the present work polymeric nanoparticles based on Poly (maleic anhydride-alt-butyl vinyl ether) 5% grafted with m-PEG (2000) and 95% grafted with 2-methoxyethanol (VAM41-PEG) were loaded with human hemoglobin (Hb) and characterized from a physicochemical point of view. The assessment of structural and functional features of the loaded Hb was performed and the effect of the introduction of different reducing agents as aimed at minimizing Hb oxidation during the nanoparticles formulation pro- cess, was also investigated. Nanoparticles possessing an average diameter of 138 ± 10 nm and physico- chemical features suitable for this kind of application were successfully obtained. Although the oxidation of the protein was not avoided during its loading into nanoparticles, the presence of acidic moi- eties in the polymeric structure is proposed to be directly involved in the protein inactivation mechanism. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction Medical interest in developing artificial oxygen carriers to be used as blood substitutes is stemming from the many limitations related to the commonly used blood transfusion. It is well known that collected blood samples present numerous drawbacks con- nected to time storage, timely availability and allogenic properties. Moreover, although current blood supply is safer than ever thanks to improved donor screening techniques and samples collection conditions, the diseases associated with blood transfusion can al- ways occur due mainly to new viruses occurrence and eventually reduction of blood donors. The potential benefits of developing artificial blood substitutes can be related to the universal compatibility, the on need availabil- ity, freedom from disease transmission, and long-term storage potential. In the past decades, many strategies based on hemoglobin (Hb) and fluorocarbons have been investigated in order to obtain artifi- cial oxygen carriers that could solve the above mentioned donor transfusion associated drawbacks. Among Hb based cellular systems, the use of polymeric nano- particles as artificial oxygen carriers presents many advantages mainly related to the feasibility of co-encapsulating reducing agents that can avoid Hb oxidation during the formulation process, and to the feasibility of easily modifying polymeric nanoparticles surfaces with antiopsonizing moieties that can impart Hb-loaded nanoparticles stealth properties (Piras et al., 2008). Avoiding Hb oxidation during nanoparticles preparation is a key issue in devel- oping Hb loaded polymeric nanoparticles. The introduction of dif- ferent reducing agents into Hb loaded nanoparticles formulation processes has been widely reported in literature; among these cat- alase, superoxide dismutase, peroxidase (Wright et al., 1996; Ter- amura et al., 2003; Napolitano, 2009), ascorbic acid, glutathione and methylene blue (Sakai et al., 1994; Sachdeva et al., 2003; Malchesky, 2010) can be cited. Poly (maleic anhydride-alt-butyl vinyl ether) 5% grafted with m-PEG (2000) and 95% grafted with 2-methoxyethanol (VAM41- PEG) is an amphiphilic synthetic polymer which has shown favor- able physical–chemical properties in the formulation of bioerodible polymeric nanostructured systems for the controlled release of high and low molecular weight active agents (Chiellini et al., 2008a,b; Piras et al., 2008). Each repeating unit of the hemiesters carries a hydrophilic ionizable acid group, a hydrophobic but potentially degradable ester residue, and an alkyl ether moiety. The polymer water solubility gradually increases under physiological conditions, thanks to the progressive carboxyl ionization and to an autocata- lytic or enzyme mediated ester hydrolysis (Woodruff et al., 1972; Vert et al., 1992). Furthermore, the presence of PEG moieties even- tually grafted onto the polymer matrix confers to the functionalised system stealth properties. The present study is aimed at investigating the maintenance of Hb structural and functional features after its loading into 0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.03.010 ⇑ Corresponding author. Tel.: +39 050 2210305; fax: +39 050 2210332. E-mail address: federica@dcci.unipi.it (F. Chiellini). 1 Alberto Dessy and Anna M. Piras contributed equally to this paper. European Journal of Pharmaceutical Sciences 43 (2011) 57–64 Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps