Nanoquímica con bacterias, biopolímeros y biofibras. Una nueva vía para el tratamiento de infecciones

Resumen

This PhD thesis is devoted to the development of new methodologies for producing biomaterials by way of the adequate integration of probiotic bacteria into polymeric matrices (collagen or bacterial cellulose). An in-depth characterization of the resulting hybrid materials in terms of their structure, mechanical properties and biological performance has demonstrated their versatility for use in real medical applications, specifically the antibiotic-free treatment of bacterial infections. The experimental results and corresponding discussions are outlined in six chapters as follow: Chapter 1 introduces some of the basic concepts of the research areas in which this thesis is framed. The concept “Biomaterial” is defined, focusing the discussion on their applicability in medicine. Subsequently, the two most important biopolymers used in this thesis, namely collagen and bacterial cellulose, are described in detail, discussing their structures, biological importance, properties and, more generally, some of their applications. These biopolymers have been used as scaffolds to develop innovative living materials (LMs), a relatively new concept of biomaterial comprising an inert matrix that incorporates living entities, usually cells. In this thesis, we have used probiotic bacteria as living entities instead. Thus, the introduction continues with a discussion of the most interesting bacterial types in the biomedical industry, differentiating between those that are beneficial to human health, known as probiotic bacteria, and pathogenic bacteria, which are known for their ability to infect or cause disease. With respect to pathogenic bacteria, we decided to focus on one of the greatest current global health threats, namely the emergence and exponential development of antibiotic-resistant bacteria. Finally, the main objectives of this thesis are clearly described. Chapter 2 describes the first example of an LM, in which self-assembled collagen fibres serve as scaffolds for probiotic bacteria and their exopolysaccharides, for the treatment of bacterial vaginosis (BV). BV is the most common infection in women of childbearing age and is characterised by an imbalance in the vaginal microbiota. BV can be considered a model of infection, with vaginal microbiota and pathogenic bacteria struggling for predominance and survival, thereby resulting in either a healthy state or infection, depending on the predominance of the former or the latter. In this chapter, we have developed a strategy to encapsulate (and protect) Lactobacillus fermentum or Lactobacillus acidophilus, two probiotics widely used in the food industry, in 3D collagen matrices. Thus, self-assembly of collagen in the presence of the probiotics resulted in the complete integration of the bacteria, which use their EPS to interact specifically with the collagen fibrils, thus leading to the formation of a LM with a good level of performance. Specifically, the probiotics incorporated into the matrix increase their viability and metabolic activity, even under adverse conditions, in comparison to free probiotics. A further important aspect is the good adherence of this biomaterial to tissues due to the presence of collagen. These aspects make this type of biomaterial a very promising alternative for treating BV with probiotics, since its greater adherence to the vagina and its prolonged activity, due to its greater stability, would prevent relapses of this infection, one of the main disadvantages of conventional BV treatments. The protocol for obtaining this biomaterial, and its uses, have been protected under the Patent Cooperation Treaty (PCT, (P18057EP00, 2020) and transferred to the company BIOSEARCH SA. The results of this chapter have also been published (Adv. Mater. Tech. 2020, 2000137). Chapter 3 moves the focus to cellulose, the other important biopolymer widely used in medicine. This chapter concerns the development of a biomaterial with two potentially antibacterial components encapsulated in the bacterial cellulose, namely a probiotic (Lactobacillus fermentum) and silver nanoparticles. For this purpose, a “double-sided” bacterial cellulose was prepared in which one side was functionalised with silver nanoparticles and the other with probiotics. The antibacterial activity of this biomaterial against Pseudomonas aeruginosa was higher than that of its cellulose, cellulose-silver and cellulose-probiotic counterparts, thus indicating a synergy between the two antibacterial components. The results of this chapter were published in the journal Molecules 2021, 26, 2848. Chapter 4 presents other interesting example of LMs formed by integrating probiotics (L. fermentum or L. gasseri) into the bacterial cellulose matrix. This protocol is based on co-culture of the probiotic and the cellulose-producing bacterium A. xylinum under conditions initially favourable for A. xylinum to obtain the cellulose matrix, and subsequently switched to optimal culture conditions for growth of the probiotic. This induces exponential growth of the probiotic, which completely invades the cellulose membrane, displacing the cellulose-producing bacteria. Importantly, this protocol avoids the costly and time-consuming purification treatment commonly required to remove the cellulose-producing bacteria. This type of biomaterial showed antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa and, more importantly, against multidrug resistant S. aureus and P. aeruginosa isolated from urine samples of real patients. Noticeably, all antibacterial assays were carried out in optimal media and culture conditions for the pathogenic bacteria, thus highlighting the great potential of this new type of biomaterial as an antibacterial agent. This biomaterial has been protected (PCT/EP2021 068166) and published in Acta Biomaterialia 2021, 124, 244. The results described in chapter 5 demonstrate the high level of performance that can be achieved upon the optimal integration of living entities into bacterial cellulose. We carried out a full dynamic study of the mechanical (rheological) properties of the biomaterial obtained after integrating the probiotic L. fermentum into bacterial cellulose and observed how proliferation of the probiotic in the cellulose network induces very significant changes in the mechanical properties of the biomaterial. In particular, we observed how the viscoelasticity of this biomaterial can be tuned by bacterial proliferation. Thus, at low probiotic density, the biomaterial consists of a gel with a lower viscoelasticity than the matrix, while massive proliferation of the probiotic in the cellulose matrix causes the development of mechanical properties typical of a solid. This transformation from gel to solid with the “simple” passage of culture time (i.e., proliferation) has not been observed in any other living material and opens up the possibility that such biomaterials can be used for in vivo 3D printing in different biomedical applications. The chapter 6 starts with the synthesis and structural characterisation of the bacterial cellulose produced by Acetobacter xylinum, both with the bacteria incorporated into the cellulose structure and after purification, once bacteria were removed. We have also explored the impact of culture conditions and drying method on the bacterial cellulose structure. This study formed the cornerstone for tackling the challenge of aligning cellulose fibres with the aim of obtaining bacterial cellulose with improved mechanical properties compared to the native form. In this regard, we have developed a novel in situ method for obtaining cellulose with a high degree of fibre alignment using modified “magnetic” A. xylinum. Culture of these bacteria in the presence of magnetic fields results in the formation of a thin cellulose sheet. A preliminary structural characterization by AFM, SAXS and WAXS suggests the appearance of anisotropy and some degree of preferential orientation of the cellulose fibres.