J. of Supercritical Fluids 56 (2011) 292–298 Contents lists available at ScienceDirect The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu Tailoring thermoresponsive microbeads in supercritical carbon dioxide for biomedical applications Eunice Costa a , Jorge de-Carvalho a , Teresa Casimiro a , Cláudia Lobato da Silva b , Maria Teresa Cidade c , Ana Aguiar-Ricardo a,∗ a REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b IBB, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal c Departamento de Ciências dos Materiais e CENIMAT, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal article info Article history: Received 1 June 2010 Received in revised form 27 September 2010 Accepted 20 October 2010 Keywords: Microbeads Supercritical carbon dioxide PNIPAAm Thermoresponsive Cross-linkers abstract The preparation of smart polymeric particles in supercritical carbon dioxide (scCO 2 ) presents many advantages for biomedical applications over conventional processes due to the easy elimination of trace contaminants rendering highly pure particles. Herein we report the successful optimization of poly(N- isopropylacrylamide) (PNIPAAm) synthesis strategy to obtain cell-sized hydrogel microbeads with defined and systematically varied mechanical properties. The effect of using different hydrophilic cross- linkers such as N,N-methylenebisacrylamide (MBAm), di(ethylene) glycol dimethacrylate (DEGDMA) and glycerol dimethacrylate (GDMA), on beads morphological, physico-chemical and mechanical properties was investigated. In agreement with a larger water uptake ability beads cross-linked with DEGDMA are more compliant than those containing MBAm or GDMA, having lower stiffness as accessed through oscillatory measurements on a rotational rheometer. Cytotoxicity assays showed that the obtained cross- linked PNIPAAm microbeads do not present any toxic effect on fibroblast cell cultures. Microbeads biocompatibility and adequate mechanical compliance enable their potential application on biomedical settings. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Hydrogels are cross-linked networks of hydrophilic poly- mers that have been widely explored for different biomedical applications due to their tunable chemical properties and three-dimensional physical structure, high water content, biocom- patibility and mechanical properties similar to natural biological tissues. These features offer great potential for their use in tis- sue engineering, biomedical implants, drug delivery systems and bionanotechnology [1,2]. Different strategies may be pursued to control the water uptake and mechanical properties of hydrogels: altering the co-monomer composition and/or cross-linking density, and changing the experimental conditions under which the poly- mer is formed [3]. Hydrogels can be in the form of macroscopic networks or confined to smaller dimensions such as microgels. Microgels have been traditionally prepared using a vast array of synthetic techniques, including photolithographic and micro- molding methods, microfluidics, modification of biopolymers with various approaches, and free radical heterogeneous polymerization in dispersion, precipitation and emulsion [4]. Many of these pro- ∗ Corresponding author. Tel.: +351 212 949 648; fax: +351 212 948 550. E-mail address: aar@dq.fct.unl.pt (A. Aguiar-Ricardo). duction routes involve an excessive use of organic solvents both in the reaction medium and in subsequent purification steps [5]. The use of supercritical carbon dioxide (scCO 2 ) as a polymerization medium for the preparation of microgels offers many advantages over conventional solvents: CO 2 is nontoxic, nonflammable, inex- pensive and readily available in high purity from a variety of sources [6]. Since it is a gas at normal pressure by simply reducing the pres- sure of the system, it is possible to easily separate the solvent from the polymer, leading to highly pure materials ideal for biomedical applications [7]. PNIPAAm is a thermoresponsive hydrogel with a low critical solution temperature (LCST) between 30 ◦ C and 32 ◦ C in an aque- ous solution, close to body temperature [8,9]. It dissolves in water below the LCST and precipitates from the aqueous solution above the LCST due to the disruption of hydrogen bonding with water and the increasing hydrophobic interactions among isopropyl groups. PNIPAAm hydrogels have been widely employed in a diverse range of biomedical applications, such as drug controlled release, enzyme immobilization and cell sheet technology due to this unique prop- erty [10–13]. In this work we intend to prepare cell-sized PNIPAAm microgels with well-defined morphology and mechanical proper- ties to be used in the development of biosensing platforms to assess cell microenvironments in tissue cultures. Studies have demon- strated that the particle size plays a key role in their adhesion 0896-8446/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2010.10.039