Introduction: Calcium ions (Ca2+) are biologically-benign cations found throughout our bodies. This feature allows us to use Ca2+ for designing biomaterials. For example, alginate gels cross-linked with Ca2+ are used in a variety of biomedical applications[1]. However, no chemically-synthesized polymer has been reported to cross-link with Ca2+ to form gels under physiological conditions, to the best of our knowledge.
In this study, we designed a novel star block copolymer with double hydrophilic block copolymer arms that can cross-link with Ca2+. We used dendritic polyester (DPE) as a star core, and block copolymers of oligo(ethylene glycol) methyl ether acrylate (OEGA) and acrylic acid (AA) as star arms[2]. Gelation behavior and biocompatibility of the star block copolymer were examined.
Materials and Methods: We first synthesized the star block copolymer. The terminal hydroxyl groups of DPE were transformed into 2-bromoisobutyryl groups to obtain DPE-Br. Using the DPE-Br as an initiator, the star arms were then polymerized by atom transfer radical polymerization (ATRP) of OEGA and, subsequently, t-butyl acrylate (tBA) to obtain DPE-g-OEGA-b-tBA. Finally, deprotection of t-butyl groups was performed to obtain DPE-g-OEGA-b-AA.
We then conducted inversed vial tests. Equal volumes of the 40 wt% star block copolymer in normal saline (NS) and 0.5 M sodium chloride (NaCl) or calcium chloride (CaCl2) in NS were mixed in glass vials. The vials were then turned upside down to determine whether gelation occurred or not.
The star block copolymer gel (0.1 mL) was finally administered into ICR mice (3 week old, male) subcutaneously (s.c.) to the posterodorsal wall or intraperitoneally (i.p.). Four individual mice were used for each condition. All the mice were sacrificed 1 week after the injections for evaluations.
Results and Discussion: The synthesis of the star block copolymer was confirmed by 1H NMR and FT-IR. Number of arms was determined to be ca. nine by 1H NMR. Weight-average molecular weight of this polymer measured by gel permeation chromatography was 3.4 × 105. The average diameter measured by dynamic light scattering was 62.2 nm.
We investigated the gelation ability of the obtained star polymer. As a result, addition of Ca2+ to the star polymer solution induced gelation, while that of Na+ did not.
We evaluated biocompatibility of this gel in vivo by s.c. and i.p. injection into mice. The gels were completely cleared from the injection sites 1 week after the injection. No mice showed decrease in body weights. We observed no pathological appearance. All the results above confirmed that the star block copolymer gel had good biocompatibility.
Conclusion: We have synthesized a novel star block copolymer with DPE core and block copolymer arms of OEGA and AA. This star block copolymer formed a gel by mixing with Ca2+. To the best of our knowledge, this is the first report on formation of a totally-synthetic gel induced by Ca2+ under a physiological condition. The obtained gel showed high biocompatibility in vivo.
We deeply thank Perstorp Japan Co., Ltd. for supplying DPE.; We deeply thank Shin-Nakamura Chemical Co., Ltd. for supplying OEGA.; We would like to thank Prof. Yamaguchi and Prof. Tsuji at The University of Tokyo for the access of DLS.
References:
[1] A.D. Augst, H.J. Kong, and D.J. Mooney, “Alginate Hydrogels as Biomaterials”, Macromol. Biosci. 2006, 6, 623.
[2] Y. Nakagawa, Y. Amano, S. Nakasako, S. Ohta, and T. Ito, “Biocompatible Star Block Copolymer Hydrogel Cross-linked with Calcium Ions”, ACS Biomater. Sci. Eng. In press.