1. Smart bionanomaterials for treatment and diagnosis of inflammatory bowel diseaseSpase Stojanov, Aleš Berlec, 2024, pregledni znanstveni članek Povzetek: Inflammatory bowel disease (IBD) encompasses a group of chronic inflammatory disorders that affect the gastrointestinal tract, with Crohn’s disease and ulcerative colitis being the primary subtypes. Diagnosis and treatment of IBD are challenging due to their unknown etiology and complex pathology. Smart bionanomaterials, which are biocompatible nanometer-sized materials that respond to external stimuli, can be used in the treatment and diagnosis of diseases. In the context of IBD, these materials can deliver drugs, primarily aminosalicylates, and corticosteroids, as well as live probiotics to the inflamed parts of the intestine, with a specific focus on the colon. The controlled release of drugs can be triggered by the conditions present in the IBD-affected intestine, such as inflammation, anaerobic environment, neutral pH, and gut microbiota. This article provides an overview of the use of smart bionanomaterials, including hydrogels, nanoparticles, nanofibers, and hybrid systems. It discusses their manufacturing process and their ability to deliver active ingredients in response to various stimuli, such as pH, temperature, reactive oxygen species, magnetic field, and biomolecules, for the treatment of IBD. We also describe the use of smart probiotics, which have been genetically engineered to recognize specific stimuli and synthesize recombinant proteins for the treatment of IBD. The qualitative or quantitative response to inflammatory stimuli can be exploited in diagnostic applications, with some examples already developed. Smart bionanomaterials offer several advantages, such as encapsulation, targeted delivery, responsiveness to stimuli, and controlled release. These features make them a valuable adjunct tool in the diagnosis and treatment of IBD.
Ključne besede: bionanomaterials, responsive materials, gut microbiota
Objavljeno v DiRROS: 17.01.2025; Ogledov: 251; Prenosov: 157
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2. New treatment approaches for Clostridioides difficile infections : alternatives to antibiotics and fecal microbiota transplantationTomaž Bratkovič, Abida Zahirović, Maruša Bizjak, Maja Rupnik, Borut Štrukelj, Aleš Berlec, 2024, pregledni znanstveni članek Povzetek: Clostridioides difficile causes a range of debilitating intestinal symptoms that may be fatal. It is particularly problematic as a hospital-acquired infection, causing significant costs to the health care system. Antibiotics, such as vancomycin and fidaxomicin, are still the drugs of choice for C. difficile infections, but their effectiveness is limited, and microbial interventions are emerging as a new treatment option. This paper focuses on alternative treatment approaches, which are currently in various stages of development and can be divided into four therapeutic strategies. Direct killing of C. difficile (i) includes beside established antibiotics, less studied bacteriophages, and their derivatives, such as endolysins and tailocins. Restoration of microbiota composition and function (ii) is achieved with fecal microbiota transplantation, which has recently been approved, with standardized defined microbial mixtures, and with probiotics, which have been administered with moderate success. Prevention of deleterious effects of antibiotics on microbiota is achieved with agents for the neutralization of antibiotics that act in the gut and are nearing regulatory approval. Neutralization of C. difficile toxins (iii) which are crucial virulence factors is achieved with antibodies/antibody fragments or alternative binding proteins. Of these, the monoclonal antibody bezlotoxumab is already in clinical use. Immunomodulation (iv) can help eliminate or prevent C. difficile infection by interfering with cytokine signaling. Small-molecule agents without bacteriolytic activity are usually selected by drug repurposing and can act via a variety of mechanisms. The multiple treatment options described in this article provide optimism for the future treatment of C. difficile infection.
Ključne besede: microbiota, immunomodulators
Objavljeno v DiRROS: 17.01.2025; Ogledov: 337; Prenosov: 156
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3. Modified vaginal lactobacilli expressing fluorescent and luminescent proteins for more effective monitoring of their release from nanofibers, safety and cell adhesionSpase Stojanov, Tina Vida Plavec, Špela Zupančič, Aleš Berlec, 2024, izvirni znanstveni članek Povzetek: Electrospun nanofibers offer a highly promising platform for the delivery of vaginal lactobacilli, providing an innovative approach to preventing and treating vaginal infections. To advance the application of nanofibers for the delivery of lactobacilli, tools for studying their safety and efficacy in vitro need to be established. In this study, fluorescent (mCherry and GFP) and luminescent (NanoLuc luciferase) proteins were expressed in three vaginal lactobacilli (Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii) and a control Lactiplantibacillus plantarum with the aim to use this technology for close tracking of lactobacilli release from nanofibers and their adhesion on epithelial cells. The recombinant proteins influenced the growth of the bacteria, but not their ability to produce hydrogen peroxide. Survival of lactobacilli in nanofibers immediately after electrospinning varied among species. Bacteria retained fluorescence upon incorporation into PEO nanofibers, which was vital for evaluation of their rapid release. In addition, fluorescent labelling facilitated efficient tracking of bacterial adhesion to Caco-2 epithelial cells, while luminescence provided important quantitative insights into bacterial attachment, which varied from 0.5 to 50% depending on the species. The four lactobacilli in dispersion or in nanofibers were not detrimental for the viability of Caco-2 cells, and did not demonstrate hemolytic activity highlighting the safety profiles of both bacteria and PEO nanofibers. To summarize, this study contributes to the development of a promising delivery system, tailored for local administration of safe vaginal lactobacilli.
Objavljeno v DiRROS: 17.01.2025; Ogledov: 236; Prenosov: 89
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4. Incorporation of recombinant proteins into extracellular vesicles by Lactococcus cremorisTina Vida Plavec, Kristina Žagar, Giulia Della Pelle, Špela Zupančič, Robert Vidmar, Aleš Berlec, 2025, izvirni znanstveni članek Povzetek: Extracellular vesicles (EVs) are nanosized lipid bilayer particles released by various cellular organisms that carry an array of bioactive molecules. EVs have diagnostic potential, as they play a role in intercellular interspecies communication, and could be applied in drug delivery. In contrast to mammalian cell-derived EVs, the study of EVs from bacteria, particularly Gram-positive bacteria, received less research attention. This study aimed to investigate the production of EVs by lactic acid bacterium Lactococcus cremoris NZ9000 and to examine the impact of recombinant protein expression on their formation and protein content. Four different recombinant proteins were expressed in L. cremoris NZ9000, in different forms of expression and combinations, and the produced EVs were isolated using the standard ultracentrifugation method. The presence of vesicular structures (50–200 nm) in the samples was confirmed by transmission electron microscopy and by flow cytometry using membrane-specific stain. Mass spectrometry analyses confirmed the presence of recombinant proteins in the EVs fraction, with amounts ranging from 13.17 to 100%, highlighting their significant incorporation into the vesicles, together with intrinsic L. cremoris NZ9000 proteins that were either more abundant in the cytoplasm (ribosomal proteins, metabolic enzymes) or present in the membrane. The presence of the most abundant lactococcal proteins in EVs fraction suggests that protein cargo-loading of EVs in L. cremoris NZ9000 is not regulated. However, our data suggests that L. cremoris NZ9000 genetically engineered to express recombinant proteins can produce EVs containing these proteins in scalable manner. As L. cremoris NZ9000 is considered safe bacterium, EVs from L. cremoris NZ9000 could have several advantages over EVs from other bacteria, implying possible biotechnological applications, e.g. in therapeutic protein delivery.
Ključne besede: Lactococcus cremoris, extracellular vesicles, recombinant proteins, delivery vehicle
Objavljeno v DiRROS: 16.01.2025; Ogledov: 292; Prenosov: 129
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