title: Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology.
abstract: Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest.
  
  
  
  The development of both natural and synthetic biomaterials for biomedical applications is a field in constant development. In this context, E. coli is a bacteria that has been widely studied for its ability to naturally produce functional biomaterials with broad biomedical uses. Despite being effective, products derived from this species contain membrane residues able to trigger a non-desired immunogenic responses. Accordingly, exploring alternative bacteria able to synthesize such biomaterials in a safe molecular environment is becoming a challenge. Thus, the present study describes a new type of functional protein-based nanomaterial free of toxic contaminants with a wide range of applications in both human and veterinary medicine.
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain. Electronic address: olivia.cano.garrido@gmail.com.
authors.forename: Olivia
authors.initials: O
authors.lastname: Cano-Garrido
authors.affiliation: Servei de Microscòpia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. Electronic address: Alejandro.Sanchez.Chardi@uab.cat.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sánchez-Chardi
authors.affiliation: Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain. Electronic address: silvia.pares@irta.cat.
authors.forename: Sílvia
authors.initials: S
authors.lastname: Parés
authors.affiliation: Department of Molecular Nanoscience and Organic Materials, Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Bellaterra, 08193 Barcelona, Spain. Electronic address: irene.giroparadell@gmail.com.
authors.forename: Irene
authors.initials: I
authors.lastname: Giró
authors.affiliation: CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain; Department of Molecular Nanoscience and Organic Materials, Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Bellaterra, 08193 Barcelona, Spain. Electronic address: witold.tatkiewicz@gmail.com.
authors.forename: Witold I
authors.initials: WI
authors.lastname: Tatkiewicz
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain. Electronic address: Neus.Ferrer@uab.cat.
authors.forename: Neus
authors.initials: N
authors.lastname: Ferrer-Miralles
authors.affiliation: CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain; Department of Molecular Nanoscience and Organic Materials, Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Bellaterra, 08193 Barcelona, Spain. Electronic address: iratera@icmab.es.
authors.forename: Imma
authors.initials: I
authors.lastname: Ratera
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy. Electronic address: antonino.natalello@unimib.it.
authors.forename: Antonino
authors.initials: A
authors.lastname: Natalello
authors.affiliation: Departament de Matemàtica Aplicada IV, Universitat Politècnica de Catalunya, Jordi Girona 1-3, 08034 Barcelona, Spain. Electronic address: rcubarsi@gmail.com.
authors.forename: Rafael
authors.initials: R
authors.lastname: Cubarsi
authors.affiliation: CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain; Department of Molecular Nanoscience and Organic Materials, Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Bellaterra, 08193 Barcelona, Spain. Electronic address: vecianaj@icmab.es.
authors.forename: Jaume
authors.initials: J
authors.lastname: Veciana
authors.affiliation: Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. Electronic address: alex.bach@icrea.cat.
authors.forename: Àlex
authors.initials: À
authors.lastname: Bach
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain. Electronic address: antoni.villaverde@uab.cat.
authors.forename: Antonio
authors.initials: A
authors.lastname: Villaverde
authors.affiliation: Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain. Electronic address: anna.aris@irta.cat.
authors.forename: Anna
authors.initials: A
authors.lastname: Arís
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain. Electronic address: elena.garcia@irta.cat.
authors.forename: Elena
authors.initials: E
authors.lastname: Garcia-Fruitós
mesh_terms: D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007778:Lactobacillus / Q000648:ultrastructure; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D010316:Particle Size; D011994:Recombinant Proteins / Q000096:biosynthesis*; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins
keywords: Endotoxin-free; Functional nanomaterials; GRAS; Lactic acid bacteria; Nanoparticles