Protein-loaded PLGA–PEG–PLGA microspheres: A tool for cell therapy

Abstract

A promising strategy to repair injured organs is possible by delivering a growth factor via poly-(d,l lactide-co-glycolide) (PLGA) microspheres; the latter are coated with adhesion molecules that serve as a support for cell delivery. At present, PLGA is not the optimal choice of polymer because of poor or incomplete protein release. The use of a more hydrophilic PLGA–PEG–PLGA (A–B–A) copolymer increases the degree of protein release. In this work, the impact of different combinations of (B) and (A) segments on the protein-release profile has been investigated. Continuous-release profiles, with no lag phases, were observed. The triblock ABA with a low molecular weight of PEG and a high molecular weight of PLGA showed an interesting release pattern with a small burst (<10% in 48 h) followed by sustained, protein release over 36 days. Incomplete protein release was found to be due to various causes: protein adsorption, protein aggregation and protein denaturation under acidic conditions. Interestingly, cell viability and cell adhesion on microspheres coated with fibronectin highlight the interest of these polymers for tissue engineering applications.

Introduction

Cell therapy carried out by grafting autologous or non-autologous cells is a promising strategy to repair injured organs (Delcroix et al., 2009, Dennis et al., 2007). However, the survival and functional state of the cells after transplantation still need to be improved (Delcroix et al., 2009, Tabata, 2000). Growth and differentiation factors may improve cell survival, cell differentiation and affect the immediate environment, thus enhancing graft integration. Nevertheless, the delivery of these factors still remains a technological challenge due to their fragile structure and their short half-life after administration. To overcome these difficulties, the growth factors can be protected in biodegradable microparticles which offer controlled and sustained release after administration (Aubert-Pouëssel et al., 2004, D’Aurizio et al., 2011).

Our group has demonstrated the interest of a tissue engineering system named Pharmacologically Active Microcarriers (PAMs) to improve grafting in the host tissue. These biodegradable particles made with poly-(d,l-lactic-co-glycolic acid) (PLGA) with an adapted size (60 μm), presenting a biomimetic surface of cell adhesion/extracellular matrix molecules, served as a support for cell administration and the programmed delivery of an appropriate protein. The combined effect of the 3D, biomimetic surface and the delivered growth factor increased cell survival and differentiation of the transported cells and also enhanced the regenerative potential of stem cells (Bouffi et al., 2010, Delcroix et al., 2011, Tatard et al., 2004, Tatard et al., 2005a). A PLGA polymer was first chosen because of its biodegradable and non-toxic nature (Fournier et al., 2003). However, low and incomplete protein release from PLGA microspheres are related to protein instability during the release period (Determan et al., 2006, Fu et al., 2000). Our group and others have shown that by introducing hydrophilic segments poly(ethylene glycol) (PEG) into hydrophobic polyesters, PLGA, protein release from PLGA–PEG–PLGA (ABA) triblock copolymer microspheres was enhanced (Kissel et al., 2002, Paillard-Giteau et al., 2010). Due to the presence of PEG segments, cross-linked biodegradable hydrogel formed upon contact with water thus favouring protein release (Li and Kissel, 1993). The PEG segment itself also promotes the stability of proteins (Kissel et al., 1996). In this regard, Kissel et al. (2002) demonstrate a complete release of lysozyme from PLGA–PEG–PLGA with a high protein loading of 5%.

Tissue engineering approaches combining 3D biomimetic systems and the sustained release of therapeutic factors represent a technological improvement for cell therapy studies. Accurate delivery of therapeutic proteins at physiological levels requires low encapsulation loading of these otherwise expensive proteins (VEGF, CNTF, GDNF) (Aubert-Pouëssel et al., 2004, Bertram et al., 2009, Boerckel et al., 2011), which accentuates protein destabilisation following polymer–protein interaction. However, most of research in the literature was focused at high protein loading (more than 3%) (Kissel et al., 2002) or focused on the in vivo bio-application where the reason of incomplete protein release was not clarified (Chen and Hu, 2011). It is therefore essential to investigate protein release from ABA copolymers with low loading (<1%) in order to elucidate protein behaviour during a long release period. Furthermore, although the co-precipitation of the stabilizer agent with the protein presents particular interest to stabilize the protein without affecting the burst effect (Paillard-Giteau et al., 2010), the association of the co-precipitation with the systematic study of the polymers was not yet evaluated. In the present study, the impact of ABA copolymer composition and molecular weight on the release profile and protein stability were studied. Lysozyme was used as a model protein because it is representative of the physical and chemical properties (isoelectric point and molecular weight) and the adsorption behaviour of therapeutic growths factors such as NGF, TGF-β3 and NT3 (Aubert-Pouëssel et al., 2002, Paillard-Giteau et al., 2010).

The adsorption of fibronectin on ABA copolymers was also evaluated to assess the capability of creating an appropriate 3D biomimetic surface with these copolymers. Fibronectin was used as a bioadhesive substance for this biomimetic surface because it enhances the attachment of various stem cells in vitro and affects their behaviour such as survival, migration and proliferation (Delcroix et al., 2009). Cell adhesion and cell survival on the microspheres with a fibronectin biomimetic surface were studied. The goal of this study is to follow the stability of the protein released from different PLGA–PEG–PLGA triblock copolymers and to create a biomimetic 3D surface by fibronectin adsorption in order to improve a tissue-engineering approach.