Specific nanoprobe design for MRI: Targeting laminin in the blood-brain barrier to follow alteration due to neuroinflammation.

Chronic neuroinflammation is characterized by increased blood-brain barrier (BBB) permeability, leading to molecular changes in the central nervous system that can be explored with biomarkers of active neuroinflammatory processes. Magnetic resonance imaging (MRI) has contributed to detecting lesions and permeability of the BBB. Ultra-small superparamagnetic particles of iron oxide (USPIO) are used as contrast agents to improve MRI observations. Therefore, we validate the interaction of peptide-88 with laminin, vectorized on USPIO, to explore BBB molecular alterations occurring during neuroinflammation as a potential tool for use in MRI. The specific labeling of NPS-P88 was verified in endothelial cells (hCMEC/D3) and astrocytes (T98G) under inflammation induced by interleukin 1ß (IL-1ß) for 3 and 24 hours. IL-1ß for 3 hours in hCMEC/D3 cells increased their co-localization with NPS-P88, compared with controls. At 24 hours, no significant differences were observed between groups. In T98G cells, NPS-P88 showed similar nonspecific labeling among treatments. These results indicate that NPS-P88 has a higher affinity towards brain endothelial cells than astrocytes under inflammation. This affinity decreases over time with reduced laminin expression. In vivo results suggest that following a 30-minute post-injection, there is an increased presence of NPS-P88 in the blood and brain, diminishing over time. Lastly, EAE animals displayed a significant accumulation of NPS-P88 in MRI, primarily in the cortex, attributed to inflammation and disruption of the BBB. Altogether, these results revealed NPS-P88 as a biomarker to evaluate changes in the BBB due to neuroinflammation by MRI in biological models targeting laminin.

Association between anti-PL7 antibodies and increased fibrotic component in patients with antisynthetase syndrome and interstitial lung disease: a cross-sectional study.

OBJECTIVE: To evaluate whether anti-PL7 and anti-PL12 autoantibodies are associated with a greater extent of the fibrotic component of ILD in ASSD patients. METHODS: Patients with ILD-ASSD who were positive for one of the following autoantibodies: anti-Jo1, anti-PL7, anti-PL12, and anti-EJ were included. Clinical manifestations, CPK levels, pulmonary function tests, and HCRT assessments were prospectively collected according to the Goh index. The fibrotic, inflammatory, and overall extension of the Goh index and DLCO were assessed by multiple linear analyses and compared between ASSD antibody subgroups. RESULTS: Sixty-six patients were included; 17 were positive for anti-Jo1 (26%), 17 for anti-PL7 (26%), 20 for anti-PL12 (30%), and 9 (14%) for anti-EJ. Patients with anti-PL7 and anti-PL12 had a more extensive fibrotic component than anti-Jo1. Anti-PL7 patients had a 7.9% increase in the fibrotic extension (cß = 7.9; 95% CI 1.863, 13.918), and the strength of the association was not modified after controlling for sex, age, and time of disease evolution (aß = 7.9; 95% CI 0.677, 15.076) and also was associated with an increase in ILD severity after adjusting for the same variables, denoted by a lower DLCO (aß = - 4.47; 95% CI - 8.919 to - 0.015). CONCLUSIONS: Anti-PL7-positive ASSD patients had more extensive fibrosis and severe ILD than the anti-Jo1 subgroup. This information is clinically useful and has significant implications for managing these patients, suggesting the need for early consideration of concurrent immunosuppressive and antifibrotic therapy.

Low-cost inertial microfluidic device for microparticle separation: A laser-Ablated PMMA lab-on-a-chip approach without a cleanroom.

Although microparticles are frequently used in chemistry and biology, their effectiveness largely depends on the homogeneity of their particle size distribution. Microfluidic devices to separate and purify particles based on their size have been developed, but many require expensive cleanroom manufacturing processes. A cost-effective, passive microfluidic separator is presented, capable of efficiently sorting and purifying particles spanning the size range of 15 µm to 40 µm. Fabricated from Polymethyl Methacrylate (PMMA) substrates using laser ablation, this device circumvents the need for cleanroom facilities. Prior to fabrication, rigorous optimization of the device's design was carried out through computational simulations conducted in COMSOL Multiphysics. To gauge its performance, chitosan microparticles were employed as a test case. The results were notably promising, achieving a precision of 96.14 %. This quantitative metric underscores the device's precision and effectiveness in size-based particle separation. This low-cost and accessible microfluidic separator offers a pragmatic solution for laboratories and researchers seeking precise control over particle sizes, without the constraints of expensive manufacturing environments. This innovation not only mitigates the limitations tied to traditional cleanroom-based fabrication but also widens the horizons for various applications within the realms of chemistry and biology.

Polyester urethane urea (PEUU) functionalization for enhanced anti-thrombotic performance: advancing regenerative cardiovascular devices through innovative surface modifications.

Introduction: Thrombogenesis, a major cause of implantable cardiovascular device failure, can be addressed through the use of biodegradable polymers modified with anticoagulating moieties. This study introduces a novel polyester urethane urea (PEUU) functionalized with various anti-platelet deposition molecules for enhanced antiplatelet performance in regenerative cardiovascular devices. Methods: PEUU, synthesized from poly-caprolactone, 1,4-diisocyanatobutane, and putrescine, was chemically oxidized to introduce carboxyl groups, creating PEUU-COOH. This polymer was functionalized in situ with polyethyleneimine, 4-arm polyethylene glycol, seleno-L-cystine, heparin sodium, and fondaparinux. Functionalization was confirmed using Fourier-transformed infrared spectroscopy and X-ray photoelectron spectroscopy. Bio-compatibility and hemocompatibility were validated through metabolic activity and hemolysis assays. The anti-thrombotic activity was assessed using platelet aggregation, lactate dehydrogenase activation assays, and scanning electron microscopy surface imaging. The whole-blood clotting time quantification assay was employed to evaluate anticoagulation properties. Results: Results demonstrated high biocompatibility and hemocompatibility, with the most potent anti-thrombotic activity observed on pegylated surfaces. However, seleno-L-cystine and fondaparinux exhibited no anti-platelet activity. Discussion: The findings highlight the importance of balancing various factors and addressing challenges associated with different approaches when developing innovative surface modifications for cardiovascular devices.

Genome-Scale Metabolic Reconstruction, Non-Targeted LC-QTOF-MS Based Metabolomics Data, and Evaluation of Anticancer Activity of Cannabis sativa Leaf Extracts.

Over the past decades, Colombia has suffered complex social problems related to illicit crops, including forced displacement, violence, and environmental damage, among other consequences for vulnerable populations. Considerable effort has been made in the regulation of illicit crops, predominantly Cannabis sativa, leading to advances such as the legalization of medical cannabis and its derivatives, the improvement of crops, and leaving an open window to the development of scientific knowledge to explore alternative uses. It is estimated that C. sativa can produce approximately 750 specialized secondary metabolites. Some of the most relevant due to their anticancer properties, besides cannabinoids, are monoterpenes, sesquiterpenoids, triterpenoids, essential oils, flavonoids, and phenolic compounds. However, despite the increase in scientific research on the subject, it is necessary to study the primary and secondary metabolism of the plant and to identify key pathways that explore its great metabolic potential. For this purpose, a genome-scale metabolic reconstruction of C. sativa is described and contextualized using LC-QTOF-MS metabolic data obtained from the leaf extract from plants grown in the region of Pesca-Boyaca, Colombia under greenhouse conditions at the Clever Leaves facility. A compartmentalized model with 2101 reactions and 1314 metabolites highlights pathways associated with fatty acid biosynthesis, steroids, and amino acids, along with the metabolism of purine, pyrimidine, glucose, starch, and sucrose. Key metabolites were identified through metabolomic data, such as neurine, cannabisativine, cannflavin A, palmitoleic acid, cannabinoids, geranylhydroquinone, and steroids. They were analyzed and integrated into the reconstruction, and their potential applications are discussed. Cytotoxicity assays revealed high anticancer activity against gastric adenocarcinoma (AGS), melanoma cells (A375), and lung carcinoma cells (A549), combined with negligible impact against healthy human skin cells.

Mechanoresponsive Drug Release from a Flexible, Tissue-Adherent, Hybrid Hydrogel Actuator.

Soft robotic technologies for therapeutic biomedical applications require conformal and atraumatic tissue coupling that is amenable to dynamic loading for effective drug delivery or tissue stimulation. This intimate and sustained contact offers vast therapeutic opportunities for localized drug release. Herein, a new class of hybrid hydrogel actuator (HHA) that facilitates enhanced drug delivery is introduced. The multi-material soft actuator can elicit a tunable mechanoresponsive release of charged drug from its alginate/acrylamide hydrogel layer with temporal control. Dosing control parameters include actuation magnitude, frequency, and duration. The actuator can safely adhere to tissue via a flexible, drug-permeable adhesive bond that can withstand dynamic device actuation. Conformal adhesion of the hybrid hydrogel actuator to tissue leads to improved mechanoresponsive spatial delivery of the drug. Future integration of this hybrid hydrogel actuator with other soft robotic assistive technologies can enable a synergistic, multi-pronged treatment approach for the treatment of disease.

Tailoring Magnetite-Nanoparticle-Based Nanocarriers for Gene Delivery: Exploiting CRISPRa Potential in Reducing Conditions.

Gene delivery has emerged as a promising alternative to conventional treatment approaches, allowing for the manipulation of gene expression through gene insertion, deletion, or alteration. However, the susceptibility of gene delivery components to degradation and challenges associated with cell penetration necessitate the use of delivery vehicles for effective functional gene delivery. Nanostructured vehicles, such as iron oxide nanoparticles (IONs) including magnetite nanoparticles (MNPs), have demonstrated significant potential for gene delivery applications due to their chemical versatility, biocompatibility, and strong magnetization. In this study, we developed an ION-based delivery vehicle capable of releasing linearized nucleic acids (tDNA) under reducing conditions in various cell cultures. As a proof of concept, we immobilized a CRISPR activation (CRISPRa) sequence to overexpress the pink1 gene on MNPs functionalized with polyethylene glycol (PEG), 3-[(2-aminoethyl)dithio]propionic acid (AEDP), and a translocating protein (OmpA). The nucleic sequence (tDNA) was modified to include a terminal thiol group and was conjugated to AEDP's terminal thiol via a disulfide exchange reaction. Leveraging the natural sensitivity of the disulfide bridge, the cargo was released under reducing conditions. Physicochemical characterizations, including thermogravimetric analysis (TGA) and Fourier-transform infrared (FTIR) spectroscopy, confirmed the correct synthesis and functionalization of the MNP-based delivery carriers. The developed nanocarriers exhibited remarkable biocompatibility, as demonstrated by the hemocompatibility, platelet aggregation, and cytocompatibility assays using primary human astrocytes, rodent astrocytes, and human fibroblast cells. Furthermore, the nanocarriers enabled efficient cargo penetration, uptake, and endosomal escape, with minimal nucleofection. A preliminary functionality test using RT-qPCR revealed that the vehicle facilitated the timely release of CRISPRa vectors, resulting in a remarkable 130-fold overexpression of pink1. We demonstrate the potential of the developed ION-based nanocarrier as a versatile and promising gene delivery vehicle with potential applications in gene therapy. The developed nanocarrier is capable of delivering any nucleic sequence (up to 8.2 kb) once it is thiolated using the methodology explained in this study. To our knowledge, this represents the first MNP-based nanocarrier capable of delivering nucleic sequences under specific reducing conditions while preserving functionality.

Enhancing Wound Healing: A Novel Topical Emulsion Combining CW49 Peptide and Lavender Essential Oil for Accelerated Regeneration and Antibacterial Protection.

Wound healing is a complex process involving blood cells, extracellular matrix, and parenchymal cells. Research on biomimetics in amphibian skin has identified the CW49 peptide from Odorrana grahami, which has been demonstrated to promote wound regeneration. Additionally, lavender essential oil exhibits anti-inflammatory and antibacterial activities. Given these considerations, we propose an innovative emulsion that combines the CW49 peptide with lavender oil. This novel formulation could serve as a potent topical treatment, potentially fostering the regeneration of damaged tissues and providing robust antibacterial protection for skin wounds. This study investigates the physicochemical properties, biocompatibility, and in vitro regenerative capacity of the active components and the emulsion. The results show that the emulsion possesses appropriate rheological characteristics for topical application. Both the CW49 peptide and lavender oil exhibit high viability in human keratinocytes, indicating their biocompatibility. The emulsion induces hemolysis and platelet aggregation, an expected behavior for such topical treatments. Furthermore, the lavender-oil emulsion demonstrates antibacterial activity against both Gram-positive and Gram-negative bacterial strains. Finally, the regenerative potential of the emulsion and its active components is confirmed in a 2D wound model using human keratinocytes. In conclusion, the formulated emulsion, which combines the CW49 peptide and lavender oil, shows great promise as a topical treatment for wound healing. Further research is needed to validate these findings in more advanced in vitro models and in vivo settings, potentially leading to improved wound-care management and novel therapeutic options for patients with skin injuries.

Three-dimensional neuroimmune co-culture system for modeling Parkinson's disease microenvironmentsin vitro.

Parkinson's disease (PD) is a complex and multifaceted neurodegenerative disorder that results from multiple environmental factors and multicellular interactions. Although several PD neuropathologies have been identified and described, the thorough understanding of PD pathophysiology and research has been largely limited by the absence of reliablein vitromodels that truly recapitulate PD microenvironments. Here, we propose a neuroimmune co-culture system that models PD neuropathologies by combining relevant multicellular interactions with environments that mimic the brain. This system is composed of: (i) 3D bioprinted cultures of mature human dopaminergic (DA) neurons grown on extracellular matrix (ECM)-derived scaffolds doped with electroconductive nanostructures, and (ii) a direct co-culture of human astrocytes and differentiated monocytes that models neuroinflammatory responses. When co-cultured in a transwell format, these two compartments recreate relevant multicellular environments that model PD pathologies after exposure to the neurotoxin A53Tα-synuclein. With immunofluorescent staining and gene expression analyses, we show that functional and mature DA 3D networks are generated within our ECM-derived scaffolds with superior performance to standard 2D cultures. Moreover, by analyzing cytokine secretion, cell surface markers, and gene expression, we define a human monocyte differentiation scheme that allows the appearance of both monocyte-derived macrophages and dendritic cell phenotypes, as well as their optimal co-culture ratios with human astrocytes to recreate synergistic neuroinflammatory responses. We show that the combined response of both compartments to A53Tα-synuclein stimulates the formation of intracellularα-synuclein aggregates, induces progressive mitochondrial dysfunction and reactive oxygen species production, downregulates the expression of synaptic, DA, and mitophagy-related genes, and promotes the initiation of apoptotic processes within the DA networks. Most importantly, these intracellular pathologies were comparable or superior to those generated with a rotenone-stimulated 2D control that represents the current standard forin vitroPD models and showed increased resilience towards these neurotoxic insults, allowing the study of disease progression over longer time periods than current models. Taken together, these results position the proposed model as a superior alternative to current 2D models for generating PD-related pathologiesin vitro.

Unlocking cellular barriers: silica nanoparticles and fullerenol conjugated cell-penetrating agents for enhanced intracellular drug delivery.

The limited delivery of cargoes at the cellular level is a significant challenge for therapeutic strategies due to the presence of numerous biological barriers. By immobilizing the Buforin II (BUF-II) peptide and the OmpA protein on magnetite nanoparticles, a new family of cell-penetrating nanobioconjugates was developed in a previous study. We propose in this study to extend this strategy to silica nanoparticles (SNPs) and silanized fullerenol (F) as nanostructured supports for conjugating these potent cell-penetrating agents. The same molecule conjugated to distinct nanomaterials may interact with subcellular compartments differently. On the obtained nanobioconjugates (OmpA-SNPs, BUF-II-PEG12-SNPs, OmpA-F, and BUF-II-PEG12-F), physicochemical characterization was performed to evaluate their properties and confirm the conjugation of these translocating agents on the nanomaterials. The biocompatibility, toxicity, and internalization capacity of nanobioconjugates in Vero cells and THP-1 cells were evaluated in vitro. Nanobioconjugates had a high internalization capacity in these cells without affecting their viability, according to the findings. In addition, the nanobioconjugates exhibited negligible hemolytic activity and a low tendency to induce platelet aggregation. In addition, the nanobioconjugates exhibited distinct intracellular trafficking and endosomal escape behavior in these cell lines, indicating their potential for addressing the challenges of cytoplasmic drug delivery and the development of therapeutics for the treatment of lysosomal storage diseases. This study presents an innovative strategy for conjugating cell-penetrating agents using silica nanoparticles and silanized fullerenol as nanostructured supports, which has the potential to enhance the efficacy of cellular drug delivery.

Breaking the clean room barrier: exploring low-cost alternatives for microfluidic devices.

Microfluidics is an interdisciplinary field that encompasses both science and engineering, which aims to design and fabricate devices capable of manipulating extremely low volumes of fluids on a microscale level. The central objective of microfluidics is to provide high precision and accuracy while using minimal reagents and equipment. The benefits of this approach include greater control over experimental conditions, faster analysis, and improved experimental reproducibility. Microfluidic devices, also known as labs-on-a-chip (LOCs), have emerged as potential instruments for optimizing operations and decreasing costs in various of industries, including pharmaceutical, medical, food, and cosmetics. However, the high price of conventional prototypes for LOCs devices, generated in clean room facilities, has increased the demand for inexpensive alternatives. Polymers, paper, and hydrogels are some of the materials that can be utilized to create the inexpensive microfluidic devices covered in this article. In addition, we highlighted different manufacturing techniques, such as soft lithography, laser plotting, and 3D printing, that are suitable for creating LOCs. The selection of materials and fabrication techniques will depend on the specific requirements and applications of each individual LOC. This article aims to provide a comprehensive overview of the numerous alternatives for the development of low-cost LOCs to service industries such as pharmaceuticals, chemicals, food, and biomedicine.

Multifunctional magnetoliposomes as drug delivery vehicles for the potential treatment of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. Therefore, development of novel technologies and strategies to treat PD is a global health priority. Current treatments include administration of Levodopa, monoamine oxidase inhibitors, catechol-O-methyltransferase inhibitors, and anticholinergic drugs. However, the effective release of these molecules, due to the limited bioavailability, is a major challenge for the treatment of PD. As a strategy to solve this challenge, in this study we developed a novel multifunctional magnetic and redox-stimuli responsive drug delivery system, based on the magnetite nanoparticles functionalized with the high-performance translocating protein OmpA and encapsulated into soy lecithin liposomes. The obtained multifunctional magnetoliposomes (MLPs) were tested in neuroblastoma, glioblastoma, primary human and rat astrocytes, blood brain barrier rat endothelial cells, primary mouse microvascular endothelial cells, and in a PD-induced cellular model. MLPs demonstrated excellent performance in biocompatibility assays, including hemocompatibility (hemolysis percentages below 1%), platelet aggregation, cytocompatibility (cell viability above 80% in all tested cell lines), mitochondrial membrane potential (non-observed alterations) and intracellular ROS production (negligible impact compared to controls). Additionally, the nanovehicles showed acceptable cell internalization (covered area close to 100% at 30 min and 4 h) and endosomal escape abilities (significant decrease in lysosomal colocalization after 4 h of exposure). Moreover, molecular dynamics simulations were employed to better understand the underlying translocating mechanism of the OmpA protein, showing key findings regarding specific interactions with phospholipids. Overall, the versatility and the notable in vitro performance of this novel nanovehicle make it a suitable and promising drug delivery technology for the potential treatment of PD.

The Impact of Yeast Encapsulation in Wort Fermentation and Beer Flavor Profile.

The food and beverage industry is constantly evolving, and consumers are increasingly searching for premium products that not only offer health benefits but a pleasant taste. A viable strategy to accomplish this is through the altering of sensory profiles through encapsulation of compounds with unique flavors. We used this approach here to examine how brewing in the presence of yeast cells encapsulated in alginate affected the sensory profile of beer wort. Initial tests were conducted for various combinations of sodium alginate and calcium chloride concentrations. Mechanical properties (i.e., breaking force and elasticity) and stability of the encapsulates were then considered to select the most reliable encapsulating formulation to conduct the corresponding alcoholic fermentations. Yeast cells were then encapsulated using 3% (w/v) alginate and 0.1 M calcium chloride as a reticulating agent. Fourteen-day fermentations with this encapsulating formulation involved a Pilsen malt-based wort and four S. cerevisiae strains, three commercially available and one locally isolated. The obtained beer was aged in an amber glass container for two weeks at 4 °C. The color, turbidity, taste, and flavor profile were measured and compared to similar commercially available products. Cell growth was monitored concurrently with fermentation, and the concentrations of ethanol, sugars, and organic acids in the samples were determined via high-performance liquid chromatography (HPLC). It was observed that encapsulation caused significant differences in the sensory profile between strains, as evidenced by marked changes in the astringency, geraniol, and capric acid aroma production. Three repeated batch experiments under the same conditions revealed that cell viability and mechanical properties decreased substantially, which might limit the reusability of encapsulates. In terms of ethanol production and substrate consumption, it was also observed that encapsulation improved the performance of the locally isolated strain.

Formulation of a novel antibacterial topical treatment based on Magnetite-Buforin-II-silver nanobioconjugates.

Community acquired infections caused by Meticillin-resistant Staphylococcus aureus (MRSA) have become a growing concern due to its impact on the world public health. This microorganism is a commonly spreading pathogen associated predominantly with skin infections and connected to other more severe conditions (septic shock, and generalized infection). The lack of highly effective antibiotics and treatments to control skin infections with S. aureus has led to the search of novel therapies using alternative agents such as antimicrobial peptides (AMPs). In order to obtain a viable administration route to counteract superficial skin infections (impetigo, abscesses, furuncles, and cellulitis), a topical formulation based on Magnetite-Buforin-II-silver nanobioconjugates as active antibacterial agents was designed by their dispersion in O/W concentrated emulsions. The prepared topical characterization indicated that O/W emulsions were stable in time, the droplets size remained within the appropriate values (∼1 µm) and their rheological properties, such as pseudoplastic and shear-thinning behavior, remained unchanged for up to 3 months. Additionally, hemolysis and platelet aggregation tests were acceptable (i.e., 14.72 ± 2.62% and 8.06 ± 2.90%, respectively) in compliance with the ISO-10993 standard. Furthermore, the treatment reduced significantly (p < 0.0001) the growth of both clinical isolated MRSA and wild Type S. aureus strains as evidenced by the contact diffusion method. These results are important in the context of proposing new alternatives that allow manage effectively the threat posed by the antibiotic resistant bacterial strains, which jeopardize the lives of thousands of people every year.

Magnetic Torus Microreactor as a Novel Device for Sample Treatment via Solid-Phase Microextraction Coupled to Graphite Furnace Atomic Absorption Spectroscopy: A Route for Arsenic Pre-Concentration.

This work studied the feasibility of using a novel microreactor based on torus geometry to carry out a sample pretreatment before its analysis by graphite furnace atomic absorption. The miniaturized retention of total arsenic was performed on the surface of a magnetic sorbent material consisting of 6 mg of magnetite (Fe3O4) confined in a very small space inside (20.1 µL) a polyacrylate device filling an internal lumen (inside space). Using this geometric design, a simulation theoretical study demonstrated a notable improvement in the analyte adsorption process on the solid extractant surface. Compared to single-layer geometries, the torus microreactor geometry brought on flow turbulence within the liquid along the curvatures inside the device channels, improving the efficiency of analyte-extractant contact and therefore leading to a high preconcentration factor. According to this design, the magnetic solid phase was held internally as a surface bed with the use of an 8 mm-diameter cylindric neodymium magnet, allowing the pass of a fixed volume of an arsenic aqueous standard solution. A preconcentration factor of up to 60 was found to reduce the typical "characteristic mass" (as sensitivity parameter) determined by direct measurement from 53.66 pg to 0.88 pg, showing an essential improvement in the arsenic signal sensitivity by absorption atomic spectrometry. This methodology emulates a miniaturized micro-solid-phase extraction system for flow-through water pretreatment samples in chemical analysis before coupling to techniques that employ reduced sample volumes, such as graphite furnace atomic absorption spectroscopy.

Assessing cellular internalization and endosomal escape abilities of novel BUFII-Graphene oxide nanobioconjugates.

Cell-penetrating agents based on functionalized nanoplatforms have emerged as a promising approach for developing more efficient and multifunctional delivery vehicles for treating various complex diseases that require reaching different intracellular compartments. Our previous work has shown that achieving full cellular coverage and high endosomal escape rates is possible by interfacing magnetite nanoparticles with potent translocating peptides such as Buforin II (BUF-II). In this work, we extended such an approach to two graphene oxide (GO)-based nanoplatforms functionalized with different surface chemistries to which the peptide molecules were successfully conjugated. The developed nanobioconjugates were characterized via spectroscopic (FTIR, Raman), thermogravimetric, and microscopic (SEM, TEM, and AFM) techniques. Moreover, biocompatibility was assessed via standardized hemocompatibility and cytotoxicity assays in two cell lines. Finally, cell internalization and coverage and endosomal escape abilities were estimated with the aid of confocal microscopy analysis of colocalization of the nanobioconjugates with Lysotracker Green®. Our findings showed coverage values that approached 100% for both cell lines, high biocompatibility, and endosomal escape levels ranging from 30 to 45% and 12-24% for Vero and THP-1 cell lines. This work provides the first routes toward developing the next-generation, carbon-based, cell-penetrating nanovehicles to deliver therapeutic agents. Further studies will be focused on elucidating the intracellular trafficking pathways of the nanobioconjugates to reach different cellular compartments.

Delivery and assessment of a CRISPR/nCas9-based genome editing system on in vitro models of mucopolysaccharidoses IVA assisted by magnetite-based nanoparticles.

Mucopolysaccharidosis IV A (MPS IVA) is a lysosomal disorder caused by mutations in the GALNS gene. Consequently, the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate accumulate in the lysosomal lumen. Although enzyme replacement therapy has shown essential advantages for the patients, several challenges remain to overcome, such as the limited impact on the bone lesion and recovery of oxidative profile. Recently, we validated a CRISPR/nCas9-based gene therapy with promising results in an in vitro MPS IVA model. In this study, we have expanded the use of this CRISPR/nCas9 system to several MPS IVA fibroblasts carrying different GALNS mutations. Considering the latent need to develop more safety vectors for gene therapy, we co-delivered the CRISPR/nCas9 system with a novel non-viral vector based on magnetoliposomes (MLPs). We found that the CRISPR/nCas9 treatment led to an increase in enzyme activity between 5 and 88% of wild-type levels, as well as a reduction in GAGs accumulation, lysosomal mass, and mitochondrial-dependent oxidative stress, in a mutation-dependent manner. Noteworthy, MLPs allowed to obtain similar results to those observed with the conventional transfection agent lipofectamine. Overall, these results confirmed the potential of CRISPR/nCas9 as a genome editing tool for treating MPS IVA. We also demonstrated the potential use of MLPs as a novel delivery system for CRISPR/nCas9-based therapies.

Small Diameter Cell-Free Tissue-Engineered Vascular Grafts: Biomaterials and Manufacture Techniques to Reach Suitable Mechanical Properties.

Vascular grafts (VGs) are medical devices intended to replace the function of a blood vessel. Available VGs in the market present low patency rates for small diameter applications setting the VG failure. This event arises from the inadequate response of the cells interacting with the biomaterial in the context of operative conditions generating chronic inflammation and a lack of regenerative signals where stenosis or aneurysms can occur. Tissue Engineered Vascular grafts (TEVGs) aim to induce the regeneration of the native vessel to overcome these limitations. Besides the biochemical stimuli, the biomaterial and the particular micro and macrostructure of the graft will determine the specific behavior under pulsatile pressure. The TEVG must support blood flow withstanding the exerted pressure, allowing the proper compliance required for the biomechanical stimulation needed for regeneration. Although the international standards outline the specific requirements to evaluate vascular grafts, the challenge remains in choosing the proper biomaterial and manufacturing TEVGs with good quality features to perform satisfactorily. In this review, we aim to recognize the best strategies to reach suitable mechanical properties in cell-free TEVGs according to the reported success of different approaches in clinical trials and pre-clinical trials.

Reduced Graphene Oxide-Extracellular Matrix Scaffolds as a Multifunctional and Highly Biocompatible Nanocomposite for Wound Healing: Insights into Characterization and Electroconductive Potential.

The development of novel regenerative technologies based on the implementation of natural extracellular matrix (ECM), or individual components of ECM combined with multifunctional nanomaterials such as graphene oxide and reduced graphene oxide, has demonstrated remarkable results in wound healing and tissue engineering. However, the synthesis of these nanocomposites involves great challenges related to maintaining the biocompatibility with a simultaneous improvement in their functionalities. Based on that, in this research we developed novel nanoengineered ECM-scaffolds formed by mixing small intestinal submucosa (SIS) with graphene oxide (GO)/reduced graphene oxide (rGO) to improve electrical conductivity while maintaining remarkable biocompatibility. For this, decellularized SIS was combined with GO to form the scaffold precursor for subsequent lyophilization, chemically crosslinking and in situ reduction. The obtained GO and rGO were characterized via Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), electrical conductivity testing and atomic force microscopy (AFM). The results confirm the suitable synthesis of GO, the effective reduction to rGO and the significant increase in the electrical conductivity (more than four orders of magnitude higher than bare GO). In addition, the graphene oxide/reduced graphene oxide-SIS scaffolds were characterized via Raman spectroscopy, FTIR, TGA, SEM, porosity assay (higher than 97.5% in all cases) and protein secondary structural analysis. Moreover, the biocompatibility of scaffolds was studied by standardized assays of hemolysis activity (less than 0.5%), platelet activation and deposition, and cell viability in Vero, HaCat and HFF-1 cells (higher than 90% for all evaluated cell lines on the different scaffolds). The obtained results confirm the remarkable biocompatibility, as supported by high hemocompatibility, low cytotoxicity and no negative impact on platelet activation and deposition. Finally, structural characteristics such as pore size and interconnectivity as well as superior cell attachment abilities also corroborated the potential of the developed nanoengineered ECM-scaffolds as a multifunctional nanoplatform for application in regenerative medicine and tissue engineering.

Novel Photo- and Thermo-Responsive Nanocomposite Hydrogels Based on Functionalized rGO and Modified SIS/Chitosan Polymers for Localized Treatment of Malignant Cutaneous Melanoma.

Melanoma is an aggressive type of skin cancer that accounts for over 75% of skin cancer deaths despite comprising less than 5% of all skin cancers. Despite promising improvements in surgical approaches for melanoma resection, the survival of undetectable microtumor residues has remained a concern. As a result, hyperthermia- and drug-based therapies have grown as attractive techniques to target and treat cancer. In this work, we aim to develop a stimuli-responsive hydrogel based on chitosan methacrylate (ChiMA), porcine small intestine submucosa methacrylate (SISMA), and doxorubicin-functionalized reduced graphene oxide (rGO-DOX) that eliminates microtumor residues from surgically resected melanoma through the coupled effect of NIR light-induced photothermal therapy and heat-induced doxorubicin release. Furthermore, we developed an in silico model to optimize heat and mass transport and evaluate the proposed chemo/photothermal therapy in vitro over melanoma cell cultures.