Conventional treatments for chronic wounds are often ineffective, thus new therapeutic approaches are needed, such as the delivery of immunomodulatory drugs that can reduce inflammation, restore immune cell function, and facilitate tissue regeneration. A potential drug for such an approach is simvastatin, which has major drawbacks including poor solubility and chemical instability. With the aim of developing a dressing for wound healing, simvastatin and an antioxidant were incorporated into alginate/poly(ethylene oxide) nanofibers by green electrospinning without the use of organic solvents, thanks to their prior encapsulation into liposomes. The composite liposome–nanofiber formulations exhibited fibrillar morphology (160–312 nm) and unprecedentedly high phospholipid and drug content (76%). Transmission electron microscopy revealed dried liposomes as bright ellipsoidal spots homogeneously distributed over the nanofibers. After nanofiber hydration, the liposomes reconstituted in two size populations (~140 and ~435 nm), as revealed by cutting-edge MADLS^® analysis. Lastly, in vitro assays demonstrated that composite liposome–nanofiber formulations are superior to liposomal formulations due to a better safety profile in keratinocytes and peripheral blood mononuclear cells. Furthermore, both formulations exhibited similarly advantageous immunomodulatory effects, measured as decreased inflammation in vitro. A synergistic combination of the two nanodelivery systems shows promise for the development of efficient dressings for chronic wound treatment.

Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6 months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an antiproliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites.

Thymoquinone (TQ), a bioactive compound found in Nigella sativa seeds, possesses diverse therapeutic properties for skin conditions. However, formulating TQ presents challenges due to its hydrophobic nature and chemical instability, which hinder its skin penetration. Transethosomes, as a formulation, offer an environment conducive to enhancing TQ’s solubility, stability, and skin permeation. To optimize TQ transethosomal formulations, we introduced a combination of ionic and nonionic surfactants, namely Tween 20 and sodium lauryl sulfate (SLS) or sodium lauroyl glutamate (SLG). Surfactants play a crucial role in stabilizing the formulation, reducing aggregation, improving biocompatibility, and minimizing potential toxicity. We fine-tuned the formulation composition and gained insights into its interfacial behavior using the Langmuir monolayer technique. This method elucidated the interfacial properties and behavior of phospholipids in ethosome and transethosome formulations. Our findings suggest that monolayer studies can serve as the initial step in selecting surfactants for nanocarrier formulations based on their interfacial dilational rheology studies. It was found that the addition of surfactant to the formulation increased the elasticity considering the capability of transethosomes to significantly decrease their radius when permeating the skin barrier. The results of the dilational rheology experiments were most relevant to drug permeation through the skin for the largest amplitude of deformation. The combination of Tween 20 and SLS efficiently modified the rheological behavior of lipids, increasing their elasticity. This conclusion was supported by in vitro studies, where formulation F2 composed of Tween 20 and SLS demonstrated the highest permeation after 24 h (300.23 µg/cm²). Furthermore, the F2 formulation showed the highest encapsulation efficiency (EE) of 94%, surpassing those of the control and ethosomal formulations. Additionally, this transethosomal formulation exhibited antimicrobial activity against S. aureus, with a zone of inhibition of 26.4 ± 0.3 mm. Importantly, we assessed the cytotoxicity of both ethosomes and transethosomes at concentrations ranging from 3.5 µM to 50 µM on HaCaT cell lines and found no cytotoxic effects compared to TQ hydroethanolic solution. These results suggest the potential safety and efficacy of TQ transethosomal formulations.

Immunological safety of nanofibers remains poorly reported within the scientific literature and lacks specific in vitro testing models distinct from those used to test nanoparticles. To address the challenges of currently used conventional setups being described in the literature, we developed a novel in vitro model for nanofiber mats immunogenicity testing, which enables standardization of tested surface area, excludes nanofiber mat edges, and ensures stable contacts of cells with nanofibers during the experiment. The effect of nanofibers was assessed on peripheral blood mononuclear cells (PBMCs) by measuring their metabolic activity using MTS cell proliferation assay, where key performance parameters, i.e. cell number, phytohemagglutinin-L (PHA-L) concentration, incubation time and cell lysis were optimized. Repeatability of results obtained with non-activated and PHA-L-activated PBMCs in contact with differently thick polycaprolactone nanofiber mats was compared using both models. Our model provided more reproducible results with lower variability, exhibiting its higher reliability and accuracy than the conventional one. Furthermore, results showed the presence of thicker mats resulted in reduced metabolic activity and PBMC proliferation without any observed cytotoxicity, providing additional insights into their non-immunogenic characteristics. The developed model enables more accurate biological assessment that can support new guidelines for in vitro nanofiber testing and formulation.

Nanofiber safety, especially immunogenicity, is important for their successful translation to clinical setting. This study provides a comprehensive evaluation of how nanofiber physical properties influence immune cells cultured on them, specifically peripheral blood mononuclear cells (PBMCs). We prepared nanofibers with a wide range of physical properties including various diameters, interfibrillar pore sizes and mat thicknesses, using four main polymers: polycaprolactone, alginate, chitosan, and zein. Our findings show that nanofiber diameters had only a marginal influence on the activity of immune cells, whereas interfibrillar nanofiber pore sizes had a significant effect, and mat thickness proved to have the greatest impact. Cells that penetrated deeper into the thick nanofiber mats ceased to proliferate but did not experience cytotoxicity. Moreover, we discovered that PBMCs penetrating the zein/PVP nanofiber mesh exhibited increased metabolic activity, indicating potential immunogenicity, whereas the other tested non-immunogenic nanofibers reduced it. To best of our knowledge, this study is the first to report on the impact of various nanofiber physical properties on in vitro immune cell behavior, thereby expanding the knowledge in the relatively unexplored field of nanofiber immunological safety. It underscores the need for rigorous preclinical nanofiber assessment and setting new standards for designing nanofiber-based biomedical products.

This study explores how nanofiber chemical composition influences human immune cell responses, a key factor in the body's defense against foreign substances. Nanofiber mats composed of polycaprolactone (PCL), alginate (ALG), chitosan (CS), and zein were incubated with both unstimulated and phytohemagglutinin-L-stimulated peripheral blood mononuclear cells (PBMCs), as well as immature and mature monocyte-derived dendritic cells (MoDCs). We evaluated viability, metabolic activity, and cytokine release of PBMCs while in MoDCs, we assessed surface marker expression, phagocytic capacity, and allogeneic T-cell stimulatory function of MoDCs. Results demonstrate that nanofiber composition significantly influenced immune cell responses. While PCL and ALG nanofibers showed no significant effects on PBMCs, those made of CS, reduced their metabolic activity, warranting caution in their use. In contrast, zein nanofibers increased both metabolic activity and pro-inflammatory cytokine release of PBMCs, suggesting their potential immunogenicity. In MoDCs, PCL and CS nanofibers impaired both their phenotypic maturation and functional performance, including antigen uptake and presentation. Zein nanofibers induced changes in MoDC surface marker expression, however their overall functionality remained intact. In contrast, ALG nanofibers exhibited the highest biocompatibility, with minimal impact on both immature and mature MoDCs. This favorable profile of ALG nanofibers supports their suitability for biomedical applications, such as tissue engineering and drug delivery. Overall, our findings underscore the importance of thorough immunological safety assessment of nanofiber-based biomaterials prior to their clinical use.