Functional Inclusion Bodies Produced in Bacteria as Naturally Occurring Nanopills for Advanced Cell Therapies

Abstract

Inclusion bodies (50-500 nm in diameter) produced in recombinant bacteria can be engineered to contain functional proteins with therapeutic potential. Upon exposure, these protein particles are efficiently internalized by mammalian cells and promote recovery from diverse stresses. Being fully biocompatible, inclusion bodies are a novel platform, as tailored nanopills, for sustained drug release in advanced cell therapies.

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.…”

“…However, we wanted to ensure that these particles still retained their ability to (i) mechanically stimulate the growth of mammalian cells when used as surface-decorating topographies in cell culture settings as described previously (García-Fruitós et al 2009;Seras-Franzoso et al 2013c;Diez-Gil et al 2010;Tatkiewicz et al 2013;Seras-Franzoso et al 2013b), and (ii) release functional proteins when internalized by mammalian cells (Liovic et al 2012;Vazquez et al 2012;Seras-Franzoso J et al 2013;Seras-Franzoso et al 2013b). The comparative analysis of cell proliferation on IB-decorated surfaces revealed similar properties of all tested IBs (Figure 7).…”

“…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.…”

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

“…These protein particles have been shown to provide positive mechanical stimuli when randomly deposited on cell culture surfaces rendering enhanced cell adhesion and proliferation in several cell lines [15;16] as well as triggering cell differentiation of mesenchymal stem cells towards osteoblasts [17]. In addition, when IBs formed by proteins with specific biological activities such as the enzymes catalase and dihydrofolate reductase, the Hsp70 chaperone, and leukemia Inhibitory factor [18], or the structural protein Keratin-14 [19] were added to the cell media of challenged cell cultures, IBs could complement missing activities by releasing sufficient amounts of soluble and functional protein. A part from this passive protein delivery IBs have recently shown able to be engineered the same sequence as the, LMW but present an extra peptide that has been described as a nuclear localization signal.…”