Hemoglobin conjugated micelles based on triblock biodegradable polymers as artificial oxygen carriers Quan Shi a, b , Yubin Huang a , Xuesi Chen a , Meng Wu c , Jing Sun a, b , Xiabin Jing a, * a State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China b Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China c Department of Materials Science and Engineering of Jilin University, Changchun 130025, People’s Republic of China article info Article history: Received 21 March 2009 Accepted 21 May 2009 Available online 26 June 2009 Keywords: Hemoglobin Artificial oxygen carrier Red blood cells Micelles abstract An artificial oxygen carrier is constructed by conjugating hemoglobin molecules to biodegradable micelles. Firstly a series of triblock copolymers (PEG–PMPC–PLA) in which the middle block contains pendant propargyl groups were synthesized and characterized. After the amphiphilic copolymer was self-assembled into core-shell micelles in aqueous solution, azidized hemoglobin molecules protected by carbon monoxide (CO) were conjugated to the micelles via click reaction between the propargyl and azido groups. The conjugation causes an increase of the micelle’s mean diameter. Maximum conjugation ratio is 250 wt% in the hemoglobin-conjugated micelles (HCMs). Oxygen-binding ability of the HCMs was demonstrated by converting the CO-binding state of the HCMs into O 2 -binding state. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Blood, which continuously delivers oxygen to, and takes carbon dioxide away from all tissues and cells, is of most essential impor- tance for human lives. The use of blood products in medical practice often meets various difficulties, such as short preservation period, immunological responses, potential infections, blood type incom- patibility, and shortage of blood supply. Research of blood substitutes has attracted great attention and rapid progress has been made in this field [1–4]. Among the blood-cell substitutes developed up to date, hemoglobin vesicles (HbV), prepared by encapsulating hemo- globin (Hb) molecules into lipid or polymeric vesicles, exhibit much better performance than chemically modified Hbs. The HbVs, with a diameter of 100–250 nm, have a layer of membrane which prevents the Hb molecules from contacting directly with the immunological system [5] or penetrating vascular wall [6,7]. It is expected to be the most promising artificial oxygen carrier [8–11], and is referred as ‘‘the third generation of blood-cell substitute’’. The matrix used for HbVs should combine good biocompatibility, non- toxicity and non-immunogenicity. In 1957, Chang [1] first prepared nylon and colloidin microcapsules. Recently, many studies were reported on liposome-encapsulated Hb (LEH) since Djordjevich and Miller established the method in 1977. PEG-modified lipid vesicles proved to have longer circulation time in vivo [12]. Another HbV system is recombinant human serum albumin (rHSA) and hemo- globin hybrids. They are constructed by non-specific binding force and have good O 2 binding ability [13–15]. In most cases, HbVs need to be modified with PEG to guarantee long storage time, blood compatibility and extended circle life [2,16–18]. Recently, a biodegradable polymer, polylactide (PLA), was more and more studied as the HbV matrix due to its favorable properties. Firstly, it is perfectly biocompatible and biodegradable, it can be finally degraded into carbon dioxide and water in the body without side effects [19,20]. PLA-based materials have been safely used in many biomedical applications [21,22]. Secondly, the structure of PLA-based material can be easily tailored to meet demands of different applica- tions. For example, PEG segment can be attached to the end of PLA to form an amphiphilic structure, which can self-assemble into micelles in aqueous solution and widely used in many biomedical fields [23,24]. Functional groups are also introduced into the backbone of PLA by copolymerization of LA and other functionalized monomers [25–27]. Through these functional groups, many bioactive molecules can be conjugated to the biodegradable polymer and endow it with certain bioactivities [28–30]. Thirdly, compared with traditional HbV matrixes, PLA-based materials are superior in mechanical properties, which lead to easier manufacturing, better stability and less cost [31]. Chang’s group first tried to prepare artificial blood cells with PLA material in 1976 [32]. Recently, they reported some approaches to nanoscale Hb encapsulation with PEG–PLA [33–35]. Usually, to achieve encapsulation, organic solvents are used to dissolve polymers and then the polymer solution is homogenized with aqueous solution of proteins by vigorous stirring or ultrasonication. * Corresponding author. Tel./fax: þ86 431 85262775. E-mail address: xbjing@ciac.jl.cn (X. Jing). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.05.082 Biomaterials 30 (2009) 5077–5085