Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates

Abstract

Abstract:Bacterial inclusion bodies (IBs) have been recently used to generate biocompatible cell culture interfaces with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on FGF-2 IB production and performance has been evaluated. FGF-2 has been overexpressed in Escherichia coli at 25 °C and 37 °C obtaining IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types.

Production of functional inclusion bodies in endotoxin-free Escherichia coli

“…this fact could be of bionanotechnological relevance if in this strain, the insoluble protein is organized as inclusion bodies (IBs). IBs, as sub-micron protein particles, are gaining relevance in biotechnology ) and biomedicine ) because of their penetrability in mammalian cells in absence of toxicity and their ability to release therapeutic proteins inside receiving cells, in either substitutive protein therapies (Liovic et al 2012;Talafova et al 2013;Vazquez et al 2012) or in tissue engineering Cano-Garrido et al 2013;Seras-Franzoso et al 2013b;Seras-Franzoso et al 2013a). Interestingly, they act as natural biomimetics of hormone-releasing secretory granules of the endocrine system (Villaverde 2012), what offers a plethora of therapeutic opportunities.…”

“…Due to their mechanical stability, the ability to penetrate mammalian cells in the absence of cellular damage and the release of functional protein, in a way similar to the release of functional hormones from amyloid repositories (Villaverde 2012), bacterial IBs became unexpectedly promising materials in drug delivery and in regenerative medicine Liovic et al 2012;Talafova et al 2013;García-Fruitós et al 2009;Seras-Franzoso et al 2012;Seras-Franzoso J et al 2013;Seras-Franzoso et al 2013c;Seras-Franzoso et al 2013b;Seras-Franzoso et al 2013a;Tatkiewicz et al 2013;Vazquez et al 2012;Villaverde et al 2012;Villaverde 2012). Although not determined quantitatively, contamination of IBs with bacterial LPS is a result of cell debris formation during cell disruption and separation (Neubauer et al 2006;Georgiou and Valax 1999).…”

Bacterial Inclusion Bodies: Discovering Their Better Half

“…Active protein is released from IBs when adhered to substrate on which cells grow (of interest in tissue engineering) [65][66][67], but also, and more efficiently, upon spontaneous internalization into mammalian cells (Figure 4) [47,68]. The high cell membrane avidity of bacterial IBs is probably supported by the amphiphilic nature of the material [64], that might promote an early anchorage to facilitate micropinocytosis.…”

“…basic fibroblast growth factor 2, they exert a dual action onto growing cells, both as topographies but also as functional agents. Such combined effect of IBs, exhibiting biological activity but also representing a scaffold, offer a promising approach to decorate surfaces which then mimic the natural extracellular matrix in artificial environments [65].…”

“…IB protein release can be verified by the physiological effect that the IB-derived soluble protein exerts in different cellular models in vitro [47,65], showing a biological impact such as reconstruction of the cytoskeleton (keratin 14) [69], cell growth recovery (dihydrofolate reductase, leukemia inhibitory factor, fibroblast growth factor) [47], reduced oxidative stress (catalase) [47] or anti-apoptotic rescuing (Hsp70) [47] and in vivo as immunostimulant (cytokines) [70]. Oral delivery of IBs in mice and intraperitoneal or intratumoral injection in zebrafish and in mice does not result in detectable signs of local or systemic toxicity [47,70].…”