Search

[Display omitted] Macrophages are known to depict two major phenotypes: classically activated macrophages (M1), associated with high production of pro-inflammatory cytokines, and alternatively activated macrophages (M2), which present an anti-inflammatory function. A precise control over M1-M2 polarization is a promising strategy in therapeutics to modulate both tissue regeneration and tumor progression processes. However, this is not a simple task as macrophages behave differently depending on the microenvironment. In agreement with this, non-consistent data have been reported regarding macrophages response to magnetic iron oxide nanoparticles (MNPs). To investigate the impact of both tissue microenvironment and MNPs properties on the obtained macrophage responses, single-core (SC) and multi-core (MC) citrate coated MNPs, are synthesized and, afterwards, loaded with a macrophage polarization trigger, IL-4. The developed MNPs are then tested in macrophages subjected to different stimuli. We demonstrate that macrophages treated with low concentrations of MNPs behave differently depending on the polarization stage independently of the concentration of iron. Moreover, we find out that MNPs size and morphology determines the effect of the IL-4 loaded MNPs on M1 macrophages, since IL-4 loaded SC MNPs favor the polarization of M1 macrophages towards M2 phenotype, while IL-4 loaded MC MNPs further stimulate the secretion of pro-inflammatory cytokines. [ABSTRACT FROM AUTHOR]

Polymeric microparticles are widely used as drug delivery platforms either alone or embedded in more complex structures for regenerative medicine. Emulsion-solvent evaporation is the most extensively used technique for microparticles preparation. Despite the apparent simplicity of this method, there is no general procedure for producing microparticles of predictable characteristics (particle size, size distribution, encapsulation efficiency, and drug loading). Hybrid systems such as neurofuzzy logic allow identifying relationships between inputs and outputs, expressing the generated mathematical models through rules in linguistic format. In this work, the relationships between the variables involved in the emulsion-solvent evaporation process and the quality parameters of PLGA microparticles as drug delivery systems were established. Neurofuzzy logic software was able to generate models of high predictability (> 85%) for the microspheres properties namely particle size, size distribution, encapsulation efficiency and drug loading. Moreover, the generated sets of IF-THEN rules allowed to dictate general guidelines to better select the PLGA microparticles formulation parameters. This approach would be of great interest as a starting point to set-up protocols for the development of PLGA microparticles obtained by emulsion-solvent evaporation for many applications. [ABSTRACT FROM AUTHOR]

One of the main challenges of cardiovascular tissue engineering is the development of bioresorbable and compliant small-diameter vascular grafts (SDVG) for patients where autologous grafts are not an option. In this work, electrospun bilayered bioresorbable SDVG based on blends of poly(L-lactic acid) (PLLA) and segmented polyurethane (PHD) were prepared and evaluated. The inner layer of these SDVG was surface-modified with heparin, following a methodology involving PHD urethane functional groups. Heparin was selected as anticoagulant agent, and also due to its ability to promote human umbilical vein endothelial cells (HUVECs) growth and to inhibit smooth muscle cells over-proliferation, main cause of neointimal hyperplasia and restenosis. Immobilized heparin was quantified and changes in SDVG microstructure were investigated through SEM. Tensile properties of the heparin-functionalized SDVG resembled those of saphenous vein. Vascular grafts were seeded with HUVECs and cultured on a flow-perfusion bioreactor to analyze the effect of heparin on graft endothelization under simulated physiological-like conditions. The analysis of endothelial cells attachment and gene expression (Real-Time PCR) pointed out that the surface functionalization with heparin successfully promoted a stable and functional endothelial cell layer. [ABSTRACT FROM AUTHOR]

The linings of the oral cavity are excellent needle-free vaccination sites, able to induce immune responses at distal sites and confer systemic protection. However, owing to the mucosal tissues' intrinsic characteristics, the design of effective antigen-delivery systems is not an easy task. In the present work, we propose to develop and characterize thermosensitive and mucoadhesive hydrogels for orotransmucosal vaccination taking advantage of artificial intelligence tools (AIT). Hydrogels of variable composition were obtained combining Pluronic® F127 (PF127), Hybrane® S1200 (HS1200) and Gantrez® AN119 (AN119) or S97 (S97). Systems were characterized in terms of physicochemical properties, adhesion capacity to mucosal tissues and antigen-like microspheres release. Additionally, polymers biocompatibility and their immune-stimulation capacity was assessed in human macrophages. Interestingly, cells treated with HS1200 exhibited a significant proliferation enhancement compared to control. The use of AIT allowed to determine the effect of each polymer on formulations properties. The proportions of PF127 and Gantrez® are mainly the factors controlling gelation temperature, mucoadhesion, adhesion work and gel strength. Meanwhile, cohesion and short-term microsphere release are dependent on the PF127 concentration. However, long-term microsphere release varies depending on the Gantrez® variety and the PF127 concentration used. Hydrogels prepared with S97 showed slower microsphere release. The use of AIT allowed to establish the conditions able to produce ternary hydrogels with immune-stimulatory properties together with adequate mucoadhesion capacity and antigen-like microspheres release. [ABSTRACT FROM AUTHOR]

Poly-3-hydroxybutyrate (PHB) is a biocompatible polymer produced by a wide variety of bacteria from different carbon sources. However, the carbon source effects on PHB properties are largely unknown. This study aimed to characterize PHB produced by Paraburkholderia xenovorans LB400 supplied with glucose (PHB g), mannitol (PHB m), or xylose (PHB x) as sole carbon sources and to evaluate their potential application as the main component of scaffolds obtained by electrospinning. The PHBs produced by strain LB400 had different molecular weights; the largest value corresponded to PHB m. The XRD-spectra revealed that PHB produced by strain LB400 from the three carbon sources are less crystalline than the commercially available polymer (PHB c). Moreover, the electrospinning process decreases even further their degree of crystallinity, which could lead to an improvement in the mechanical properties of the polymers. Relevantly, PHB x -microfibers exhibited mechanical characteristics similar to those of human skin. None of the scaffolds made of PHBs from strain LB400 grown in different carbon sources showed adverse effects on fibroblast cell growth. Thus, modifying the sugar used as the carbon source may be useful to tune the structural properties of PHB and its performance as a component of electrospun scaffolds, which may better fit specific biomedical applications. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Nanostructured lipid carriers (NLC) are biocompatible and biodegradable nanoscale systems with extensive application for controlled drug release. However, the development of optimal nanosystems along with a reproducible manufacturing process is still challenging. In this study, a two-step experimental design was performed and databases were successfully modelled using Artificial Intelligence techniques as an innovative method to get optimal, reproducible and stable NLC. The initial approach, including a wide range of values for the different variables, was followed by a second set of experiments with variable values in a narrower range, more suited to the characteristics of the system. NLC loaded with rifabutin, a hydrophobic drug model, were produced by hot homogenization and fully characterized in terms of particle size, size distribution, zeta potential, encapsulation efficiency and drug loading. The use of Artificial Intelligence tools has allowed to elucidate the key parameters that modulate each formulation property. Stable nanoparticles with low sizes and polydispersions, negative zeta potentials and high drug loadings were obtained when the proportion of lipid components, drug, surfactants and stirring speed were optimized by FormRules® and INForm®. The successful application of Artificial Intelligence tools on NLC formulation optimization constitutes a pioneer approach in the field of lipid nanoparticles. [ABSTRACT FROM AUTHOR]

Lyophilization is often employed to transform nanoparticle suspensions to stable solid forms. This work proposed Neurofuzzy Logic (NFL) to better understand the lyophilization process of Nanostructured Lipid Carriers' (NLCs) dispersions and the carbohydrate cryoprotectants' (CPs) performance in these processes. NLCs were produced by hot homogenization, frozen at different speeds, and lyophilized using several CPs at variable concentrations. NLCs were characterized, and results were expressed as increase in particle size (Δ size), polydispersity (Δ PdI), and zeta potential (Δ ZP) of lyophilized powders (LP) regarding initial dispersions. CPs were classified according to their molecular weights (MW), and the osmolarities (Π) of CPs solutions were also determined. Databases obtained were finally modelled through FormRules® (Intelligensys Ltd., Kirkwall, Scotland, UK), an NFL software. NFL models revealed that CPs' MW determines the optimal freezing conditions and CPs' proportions. The knowledge generated allowed the establishment of a traffic light system intended to successfully select and apply sugars for nanoparticles lyophilization. [ABSTRACT FROM AUTHOR]

Numerous therapeutic strategies have been developed for osteoarthritis (OA) management, including intra-articular (IA) injections. The ideal IA formulation should control cartilage degradation and restore synovial fluid viscosity. To this end, we propose to combine thermo-sensitive polymers (poloxamers) with hyaluronic acid (HA) to develop suitable beta-lapachone (βLap) loaded IA formulations. The development of IA formulations with these components entails several difficulties: low βLap solubility, unknown βLap therapeutic dose and the bonded commitment of easy administration and viscosupplementation. An optimized formulation was designed using artificial intelligence tools based on the experimental results of a wide variety of hydrogels and its therapeutic capacity was evaluated on an ex vivo OA model. The formulation presented excellent rheological properties and significantly decreased the secretion of degradative (MMP13) and pro-inflammatory (CXCL8) molecules. Therefore, the developed formulation is a promising candidate for OA treatment restoring the synovial fluid rheological properties while decreasing inflammation and cartilage degradation. • βLap loaded poloxamer+hyaluronic acid hydrogels were designed for osteoarthritis. • Artificial intelligence allowed to stablish the suitable composition of hydrogels. • The optimized hydrogel was injectable and achieved adequate viscosupplementation. • Adequate βLap controlled release was observed on the optimized hydrogel. • The formulation restored synovial fluid viscosity and decreased tissue degradation. [ABSTRACT FROM AUTHOR]

Oral anti-mycobacterial treatment of Crohn's disease (CD) is limited by the low aqueous solubility of drugs, along with the altered gut conditions of patients, making uncommon their clinical use. Hence, the aim of the present work is focused on the in vitro evaluation of rifabutin (RFB)-loaded Nanostructured lipid carriers (NLC), in order to solve limitations associated to this therapeutic approach. RFB-loaded NLC were prepared by hot homogenization and characterized in terms of size, polydispersity, surface charge, morphology, thermal stability, and drug payload and release. Permeability across Caco-2 cell monolayers and cytotoxicity and uptake in human macrophages was also determined. NLC obtained were nano-sized, monodisperse, negatively charged, and spheroidal-shaped, showing a suitable drug payload and thermal stability. Furthermore, the permeability profile, macrophage uptake and selective intracellular release of RFB-loaded NLC, guarantee an effective drug dose administration to cells. Outcomes suggest that rifabutin-loaded NLC constitute a promising strategy to improve oral anti-mycobacterial therapy in Crohn's disease. [ABSTRACT FROM AUTHOR]

Macrophages play a very important role in the wound healing process and in the inflammatory reactions against implanted materials. They polarize into two major phenotypes, pro-inflammatory (M1) and anti-inflammatory (M2) that participate in different biological processes. The aim of this work was to evaluate whether surface grafting of polypropylene (PP) and polytetrafluoroethylene (PTFE) with amino groups, claimed to improve cell adhesion, may modify macrophages polarization. Gamma radiation was applied to obtain PP films with 3%, 6% and 15% nitrogen (N) and PTFE films with 8% N. Amino-functionalized films presented higher Young's Modulus and adsorbed more serum albumin (BSA) than fibrinogen (FB). Balb/3T3fibroblast cells and human macrophages showed enhanced adhesion on functionalized films after 2 h of culture. Macrophages polarization was monitored by means of cytokines secretion for both M1 and M2 markers (IL-6, IL-10, IL-12, IL-1β, INF-γ, TNF-α and metalloproteinase-8). Cytokine levels related to acute inflammatory phase, especially TNF-α, were lower on amino-functionalized films, although the production of anti-inflammatory markers (IL-10) was not different. Overall, amino-functionalization attenuates the secretion of pro-inflammatory cytokines, which may be useful for the correct design of wound healing materials. Unlabelled Image • PP and PTFE with tunable content in amino groups were prepared. • Amino-functionalized films showed lower water contact angle. • Amino-functionalized films were cytocompatible with macrophages and fibroblasts. • Macrophages adhered faster to amino-functionalized films. • Expression of IL-12, IL-1β, TNF-α, IFN-γ, IL-6, IL-10 and MMP-8 was monitored. [ABSTRACT FROM AUTHOR]

The aim of this study was to design and evaluate novel cyclodextrin (CD)-based aggregate formulations to efficiently deliver nepafenac topically to the eye structure, to treat inflammation and increase nepafenac levels in the posterior segment, thus attenuating the response of inflammatory mediators. The physicochemical properties of nine aggregate formulations containing nepafenac/γ-CD/hydroxypropyl-β (HPβ)-CD complexes as well as their rheological properties, mucoadhesion, ocular irritancy, corneal and scleral permeability, and anti-inflammatory activity were investigated in detail. The results were compared with a commercially available nepafenac suspension, Nevanac® 3 mg/mL. All formulations showed microparticles, neutral pH, and negative zeta potential (–6 to –27 mV). They were non-irritating and nontoxic and showed high permeation through bovine sclera. Formulations containing carboxymethyl cellulose (CMC) showed greater anti-inflammatory activity, even higher than the commercial formulation, Nevanac® 0.3%. The optimized formulations represent an opportunity for topical instillation of drugs to the posterior segment of the eye. [ABSTRACT FROM AUTHOR]

Local treatment of Inflammatory Bowel Disease (IBD) has been pointed out to be a novel therapeutic approach with several advantages when compared to conventional therapies. However, the development of systems able to fulfil the requirements of this administration route is not an easy task. The present work suggests the utilization of Artificial Intelligence Tools (AIT) as an instrument to understand polymer-polymer interactions towards obtaining thermosensitive hydrogels suitable for protein rectal administration in IBD. Enemas composed by Pluronic® F127 and F68 and Methocel® K4M were developed and characterised. Two experimental designs were carried out in order to determine the effect of each polymer on their texturometric and rheological behaviour. Using the results of the first experimental design we can justify the inclusion of each raw material PF127, PF68 and MK4M in the formulation and conclude that a compromise solution is necessary to obtain thermosensitive hydrogels of the required properties. The results of the second experimental design allowed concluding that PF127 ruled mainly syringeability and bioadhesion work. On the other hand, PF68 modulated principally gelation temperature, viscosity and protein release from hydrogel matrix. Finally, MK4M influenced bioadhesiveness and mostly determined viscosity. AIT also allowed delimiting the design space to produce easy administrable and highly bioadhesive enemas that undergo fast sol-gel transitions at body temperature. • Artificial intelligence tools allowed to get tailor made protein delivery hydrogels. • Networks smart design elucidated their composition-properties relation. • Thermosensitive hydrogels ensure bioadhesion and immediate drug release. • Systems within the design space fulfil all the desired characteristics. [ABSTRACT FROM AUTHOR]

Bovine mastitis is a widespread infectious disease with a significant economic burden, accounting for 80 % of the antibiotic usage in dairy animals. In recent years, extensive research has focused on using biomimetic approaches such as probiotics, bacteriocins, bacteriophages, or phytochemicals as potential alternatives to antibiotics. The local administration of therapeutic molecules through the intramammary route is one of the most commonly strategies to manage bovine mastitis. This review highlights the most important findings in this field and discusses their local application in mastitis therapy. In contrast to antibiotics, the proposed alternatives are not limited to promote bacterial death but consider other factors associated to the host microenvironments. To this end, the proposed biomimetic strategies can modulate different stages of infection by modifying the local microbiota, preventing oxidative stress, reducing bacterial adhesion to epithelial cells, modulating the immune response, or mediating the inflammatory process. Numerous in vitro studies support the antimicrobial, antibiofilm or antioxidant properties of these alternatives. However, in vivo studies incorporating these components within pharmaceutical formulations with potential clinical application are limited. The development of secure, stable, and effective drug delivery systems based on the proposed options is necessary to achieve real alternatives to antibiotics in the clinic. [Display omitted] • Non-antibiotic strategies are being considered in bovine mastitis to reduce resistance. • Probiotics, bacteriocins, phytochemicals and bacteriophages are the most studied. • In vivo testing in bovine hosts is limited, hindering their progress to the clinic. • Intramammary pharmaceutical formulations including them need to be developed. [ABSTRACT FROM AUTHOR]

One of the main concerns in oligonucleotide-based therapeutics is achieving a successful cell targeting while avoiding drug degradation and clearance. Nanoparticulated drug delivery systems have emerged as a way of overcoming these issues. Among them, membrane-coated nanoparticles are of increasing relevance mainly due to their enhanced cellular uptake, immune evasion and biocompatibility. In this study, we designed and elaborated a simple and highly tuneable biomimetic drug delivery nanosystem based on a polymeric core surrounded by extracellular vesicles (EVs)-derived membranes. This strategy should allow the nanosystems to benefit from the properties conferred by the membrane proteins present in EVs membrane, key paracrine mediators. The developed systems were able to successfully encapsulate the required oligonucleotides. Also, their characterisation through already well standardised methods (dynamic light scattering, transmission electron microscopy and nanoparticle tracking analysis) and by fluorescence cross-correlation spectroscopy (FCCS) showed the desired core-shell structure. The cellular uptake using different cell types further confirmed the coating though an enhancement in cell internalisation of the developed biomimetic nanoparticles. This study brings up new possibilities for GapmeR delivery as it might be a base for the development of new delivery systems for gene therapy. [Display omitted] • Biomimetic EVs membrane-coated PLGA nanoparticles (BMNPs) were obtained. • FCCS confirm the coating and core-shell structure of the BMNPs. • BMNPs show better stability in physiological medium than naked polymeric cores. • BMNPs were able to efficiently load oligonucleotides. • BMNPs show enhanced cellular uptake and intracellular delivery. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is increasingly prevalent and current therapies are not completely effective. Mesenchymal stem cells are emerging as a promising therapeutic option. Here, the effect of local hydrogel application loaded with conditioned medium (CM) from human uterine cervical stem cells (hUCESC-CM) in an experimental acute colitis mice model has been evaluated. Colitis induction was carried out in C57BL/6 mice by dissolving dextran sulfate sodium (DSS) in drinking water for nine days. Ulcers were treated by rectal administration of either mesalazine (as positive control) or a mucoadhesive and thermosensitive hydrogel loaded with hUCESC-CM (H-hUCESC-CM). Body weight changes, colon length, and histopathological analysis were evaluated. In addition, pro-inflammatory TNF-α, IL-6, and IFN-γ mRNA levels were measured by qPCR. Treatment with H-hUCESC-CM inhibited body weight loss and colon shortening and induced a significant decrease in colon mucosa degeneration, as well as TNF-α, IFN-γ, and IL-6 mRNA levels. Results indicate that H-hUCESC-CM effectively alleviated DSS-induced colitis in mice, suggesting that H-hUCESC-CM may represent an attractive cell-free therapy for local treatment of IBD. [ABSTRACT FROM AUTHOR]

Blood-derived concentrated growth factors (CGFs) represent a novel autologous biomaterial with promising applications in regenerative medicine. Angiogenesis is a key factor in tissue regeneration, but the role played by CGFs in vessel formation is not clear. The purpose of this study was to characterize the angiogenic properties of CGFs by evaluating the effects of its soluble factors and cellular components on the neovascularization in an in vitro model of angiogenesis. CGF clots were cultured for 14 days in cell culture medium; after that, CGF-conditioned medium (CGF-CM) was collected, and soluble factors and cellular components were separated and characterized. CGF-soluble factors, such as growth factors (VEGF and TGF-β1) and matrix metalloproteinases (MMP-2 and -9), were assessed by ELISA. Angiogenic properties of CGF-soluble factors were analyzed by stimulating human cultured endothelial cells with increasing concentrations (1%, 5%, 10%, or 20%) of CGF-CM, and their effect on cell migration and tubule-like formation was assessed by wound healing and Matrigel assay, respectively. The expression of endothelial angiogenic mediators was determined using qRT-PCR and ELISA assays. CGF-derived cells were characterized by immunostaining, qRT-PCR and Matrigel assay. We found that CGF-CM, consisting of essential pro-angiogenic factors, such as VEGF, TGF-β1, MMP-9, and MMP-2, promoted endothelial cell migration; tubule structure formation; and endothelial expression of multiple angiogenic mediators, including growth factors, chemokines, and metalloproteinases. Moreover, we discovered that CGF-derived cells exhibited features such as endothelial progenitor cells, since they expressed the CD34 stem cell marker and endothelial markers and participated in the neo-angiogenic process. In conclusion, our results suggest that CGFs are able to promote endothelial angiogenesis through their soluble and cellular components and that CGFs can be used as a biomaterial for therapeutic vasculogenesis in the field of tissue regeneration. [ABSTRACT FROM AUTHOR]

Corneal wound healing after a trauma or a chemical injury has been shown to correlate with antioxidant levels at the ocular surface. However, ocular bioavailability of efficient antioxidants (e.g. ferulic acid) after topical administration is limited by their poor solubility, low stability and short residence time. The aim of this work was to formulate ferulic acid in a nanocomposite platform composed of nanogels and micelles for efficient delivery to cornea. Solubility enhancement factor of ferulic acid was found to be equal to 1.9 ± 0.3 and 3.4 ± 0.3 for 50 and 100 mg/ml Pluronic® F68 micellar solutions. Hyaluronan was added to blank and ferulic acid loaded micelles, and then cross-linked with ε-polylysine. Hyaluronan nanogels showed dimensions of ~300 nm with positive zeta potential values. The formulations were characterized in terms of rheological behavior, biocompatibility, wound healing properties, ferulic acid release pattern and penetration into excised bovine corneas. In comparison to Pluronic® micelles that released ferulic acid rapidly, micelle-nanogel composites sustained the release up to 2 days. Furthermore, the micelle-nanogel formulation favored in vitro wound closure promoting fibroblasts growth and ex vivo accumulation of ferulic acid into both healthy and damaged corneas (>100 µg/cm2). [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are present in all organs and tissues, playing a well-known function in tissue regeneration. However, there is also evidence indicating a broader role of MSCs in tissue homeostasis. In vivo studies have shown MSC paracrine mechanisms displaying proliferative, immunoregulatory, anti-oxidative, or angiogenic activity. In addition, recent studies also demonstrate that depletion and/or dysfunction of MSCs are associated with several systemic diseases, such as lupus, diabetes, psoriasis, and rheumatoid arthritis, as well as with aging and frailty syndrome. In this review, we hypothesize about the role of MSCs as keepers of tissue homeostasis as well as modulators in a variety of inflammatory and degenerative systemic diseases. This scenario opens the possibility for the use of secretome-derived products from MSCs as new therapeutic agents in order to restore tissue homeostasis, instead of the classical paradigm "one disease, one drug". [ABSTRACT FROM AUTHOR]