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.

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.

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.

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.

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.

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.

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.

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.

Evaluating the Impact of Thermal Processing on the Anti-Inflammatory Activity of Non-Centrifugal Cane Sugar: Implications on Cytokine Secretion and TLR4 Signaling.

Plant-derived products have gained considerable attention as inflammation modulators given the wide variety of anti-inflammatory phytochemicals reported to be present in plants and their limited side effects in vivo during prolonged exposure periods. Non-centrifugal cane sugar (NCS) has been identified as a promising sugarcane-derived product due to its high polyphenolic composition and antioxidant potential, but its incorporations into nutraceuticals and other relevant products of biomedical interest has been limited by the ample composition-wise variability resulting from extreme and loosely controlled processing conditions. Here, we assessed the effect of reducing thermal exposure during NCS processing on the retained polyphenolic profiles, as well as on their antioxidant and anti-inflammatory activities. Specifically, we proposed two modified NCS production methods that reduce exposure to unwanted thermal processing conditions by 1) limiting the employed temperatures through vacuum-aided dehydration and 2) by reducing exposure time through refractance window evaporation. By comparing the modified NCS products with traditional NCS, we showed that the proposed process strategies yield enhanced polyphenolic profiles, as evidenced by the results of the Folin-Ciocalteu polyphenol quantification method and the components identification by HPLC coupled to mass spectrometry. Although these compositional differences failed to impact the antioxidant profiles and cytocompatibility of the products, they showed an enhanced anti-inflammatory potential, given their superior modulation capacity of inflammatory cytokine secretion in both systemic and neuroinflammatory scenarios in vitro. Moreover, we showed that both modified NCS products interfere with TLR4 signaling in human monocytes to a significantly greater extent than traditional NCS. However, the anti-inflammatory effect of NCS produced under window refractance evaporation was slightly superior than under vacuum-aided dehydration, demonstrating that reducing exposure time to high temperatures is likely more effective than reducing the operation temperature. Overall, these findings demonstrated that limiting thermal exposure is beneficial for the development of NCS-based natural products with superior anti-inflammatory potential, which can be further exploited in the rational design of more potent nutraceuticals for potentially preventing chronic inflammatory diseases.

Rational Discovery of Antimicrobial Peptides by Means of Artificial Intelligence.

Antibiotic resistance is a worldwide public health problem due to the costs and mortality rates it generates. However, the large pharmaceutical industries have stopped searching for new antibiotics because of their low profitability, given the rapid replacement rates imposed by the increasingly observed resistance acquired by microorganisms. Alternatively, antimicrobial peptides (AMPs) have emerged as potent molecules with a much lower rate of resistance generation. The discovery of these peptides is carried out through extensive in vitro screenings of either rational or non-rational libraries. These processes are tedious and expensive and generate only a few AMP candidates, most of which fail to show the required activity and physicochemical properties for practical applications. This work proposes implementing an artificial intelligence algorithm to reduce the required experimentation and increase the efficiency of high-activity AMP discovery. Our deep learning (DL) model, called AMPs-Net, outperforms the state-of-the-art method by 8.8% in average precision. Furthermore, it is highly accurate to predict the antibacterial and antiviral capacity of a large number of AMPs. Our search led to identifying two unreported antimicrobial motifs and two novel antimicrobial peptides related to them. Moreover, by coupling DL with molecular dynamics (MD) simulations, we were able to find a multifunctional peptide with promising therapeutic effects. Our work validates our previously proposed pipeline for a more efficient rational discovery of novel AMPs.

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.

Blood-Vessel-Inspired Hierarchical Trilayer Scaffolds: PCL/Gelatin-Driven Protein Adsorption and Cellular Interaction.

Fabrication of scaffolds with hierarchical structures exhibiting the blood vessel topological and biochemical features of the native extracellular matrix that maintain long-term patency remains a major challenge. Within this context, scaffold assembly using biodegradable synthetic polymers (BSPs) via electrospinning had led to soft-tissue-resembling microstructures that allow cell infiltration. However, BSPs fail to exhibit the sufficient surface reactivity, limiting protein adsorption and/or cell adhesion and jeopardizing the overall graft performance. Here, we present a methodology for the fabrication of three-layered polycaprolactone (PCL)-based tubular structures with biochemical cues to improve protein adsorption and cell adhesion. For this purpose, PCL was backbone-oxidized (O-PCL) and cast over a photolithography-manufactured microgrooved mold to obtain a bioactive surface as demonstrated using a protein adsorption assay (BSA), Fourier transform infrared spectroscopy (FTIR) and calorimetric analyses. Then, two layers of PCL:gelatin (75:25 and 95:5 w/w), obtained using a novel single-desolvation method, were electrospun over the casted O-PCL to mimic a vascular wall with a physicochemical gradient to guide cell adhesion. Furthermore, tensile properties were shown to withstand the physiological mechanical stresses and strains. In vitro characterization, using L929 mouse fibroblasts, demonstrated that the multilayered scaffold is a suitable platform for cell infiltration and proliferation from the innermost to the outermost layer as is needed for vascular wall regeneration. Our work holds promise as a strategy for the low-cost manufacture of next-generation polymer-based hierarchical scaffolds with high bioactivity and resemblance of ECM's microstructure to accurately guide cell attachment and proliferation.

Predicting target-ligand interactions with graph convolutional networks for interpretable pharmaceutical discovery.

Drug Discovery is an active research area that demands great investments and generates low returns due to its inherent complexity and great costs. To identify potential therapeutic candidates more effectively, we propose protein-ligand with adversarial augmentations network (PLA-Net), a deep learning-based approach to predict target-ligand interactions. PLA-Net consists of a two-module deep graph convolutional network that considers ligands' and targets' most relevant chemical information, successfully combining them to find their binding capability. Moreover, we generate adversarial data augmentations that preserve relevant biological backgrounds and improve the interpretability of our model, highlighting the relevant substructures of the ligands reported to interact with the protein targets. Our experiments demonstrate that the joint ligand-target information and the adversarial augmentations significantly increase the interaction prediction performance. PLA-Net achieves 86.52% in mean average precision for 102 target proteins with perfect performance for 30 of them, in a curated version of actives as decoys dataset. Lastly, we accurately predict pharmacologically-relevant molecules when screening the ligands of ChEMBL and drug repurposing Hub datasets with the perfect-scoring targets.

Microfluidic Synthesis and Purification of Magnetoliposomes for Potential Applications in the Gastrointestinal Delivery of Difficult-to-Transport Drugs.

Magnetite nanoparticles (MNPs) have gained significant attention in several applications for drug delivery. However, there are some issues related to cell penetration, especially in the transport of cargoes that show limited membrane passing. A widely studied strategy to overcome this problem is the encapsulation of the MNPs into liposomes to form magnetoliposomes (MLPs), which are capable of fusing with membranes to achieve high delivery rates. This study presents a low-cost microfluidic approach for the synthesis and purification of MLPs and their biocompatibility and functional testing via hemolysis, platelet aggregation, cytocompatibility, internalization, and endosomal escape assays to determine their potential application in gastrointestinal delivery. The results show MLPs with average hydrodynamic diameters ranging from 137 ± 17 nm to 787 ± 45 nm with acceptable polydispersity index (PDI) values (below 0.5). In addition, we achieved encapsulation efficiencies between 20% and 90% by varying the total flow rates (TFRs), flow rate ratios (FRRs), and MNPs concentration. Moreover, remarkable biocompatibility was attained with the obtained MLPs in terms of hemocompatibility (hemolysis below 1%), platelet aggregation (less than 10% with respect to PBS 1×), and cytocompatibility (cell viability higher than 80% in AGS and Vero cells at concentrations below 0.1 mg/mL). Additionally, promising delivery results were obtained, as evidenced by high internalization, low endosomal entrapment (AGS cells: PCC of 0.28 and covered area of 60% at 0.5 h and PCC of 0.34 and covered area of 99% at 4 h), and negligible nuclear damage and DNA condensation. These results confirm that the developed microfluidic devices allow high-throughput production of MLPs for potential encapsulation and efficient delivery of nanostructured cell-penetrating agents. Nevertheless, further in vitro analysis must be carried out to evaluate the prevalent intracellular trafficking routes as well as to gain a detailed understanding of the existing interactions between nanovehicles and cells.

Computational Characterization of Mechanical, Hemodynamic, and Surface Interaction Conditions: Role of Protein Adsorption on the Regenerative Response of TEVGs.

Currently available small diameter vascular grafts (<6 mm) present several long-term limitations, which has prevented their full clinical implementation. Computational modeling and simulation emerge as tools to study and optimize the rational design of small diameter tissue engineered vascular grafts (TEVG). This study aims to model the correlation between mechanical-hemodynamic-biochemical variables on protein adsorption over TEVG and their regenerative potential. To understand mechanical-hemodynamic variables, two-way Fluid-Structure Interaction (FSI) computational models of novel TEVGs were developed in ANSYS Fluent 2019R3® and ANSYS Transient Structural® software. Experimental pulsatile pressure was included as an UDF into the models. TEVG mechanical properties were obtained from tensile strength tests, under the ISO7198:2016, for novel TEVGs. Subsequently, a kinetic model, linked to previously obtained velocity profiles, of the protein-surface interaction between albumin and fibrinogen, and the intima layer of the TEVGs, was implemented in COMSOL Multiphysics 5.3®. TEVG wall properties appear critical to understand flow and protein adsorption under hemodynamic stimuli. In addition, the kinetic model under flow conditions revealed that size and concentration are the main parameters to trigger protein adsorption on TEVGs. The computational models provide a robust platform to study multiparametrically the performance of TEVGs in terms of protein adsorption and their regenerative potential.

Highly Efficient Synthesis of Type B Gelatin and Low Molecular Weight Chitosan Nanoparticles: Potential Applications as Bioactive Molecule Carriers and Cell-Penetrating Agents.

Gelatin and chitosan nanoparticles have been widely used in pharmaceutical, biomedical, and nanofood applications due to their high biocompatibility and biodegradability. This study proposed a highly efficient synthesis method for type B gelatin and low-molecular-weight (LMW) chitosan nanoparticles. Gelatin nanoparticles (GNPs) were synthesized by the double desolvation method and the chitosan nanoparticles (CNPs) by the ionic gelation method. The sizes of the obtained CNPs and GNPs (373 ± 71 nm and 244 ± 67 nm, respectively) and zeta potential (+36.60 ± 3.25 mV and -13.42 ± 1.16 mV, respectively) were determined via dynamic light scattering. Morphology and size were verified utilizing SEM and TEM images. Finally, their biocompatibility was tested to assure their potential applicability as bioactive molecule carriers and cell-penetrating agents.

Design and Manufacture of a Low-Cost Microfluidic System for the Synthesis of Giant Liposomes for the Encapsulation of Yeast Homologues: Applications in the Screening of Membrane-Active Peptide Libraries.

The discovery of new membrane-active peptides (MAPs) is an area of considerable interest in modern biotechnology considering their ample applicability in several fields ranging from the development of novel delivery vehicles (via cell-penetrating peptides) to responding to the latent threat of antibiotic resistance (via antimicrobial peptides). Different strategies have been devised for such discovery process, however, most of them involve costly, tedious, and low-efficiency methods. We have recently proposed an alternative route based on constructing a non-rationally designed library recombinantly expressed on the yeasts' surfaces. However, a major challenge is to conduct a robust and high-throughput screening of possible candidates with membrane activity. Here, we addressed this issue by putting forward low-cost microfluidic platforms for both the synthesis of Giant Unilamellar Vesicles (GUVs) as mimicking entities of cell membranes and for providing intimate contact between GUVs and homologues of yeasts expressing MAPs. The homologues were chitosan microparticles functionalized with the membrane translocating peptide Buforin II, while intimate contact was through passive micromixers with different channel geometries. Both microfluidic platforms were evaluated both in silico (via Multiphysics simulations) and in vitro with a high agreement between the two approaches. Large and stable GUVs (5-100 µm) were synthesized effectively, and the mixing processes were comprehensively studied leading to finding the best operating parameters. A serpentine micromixer equipped with circular features showed the highest average encapsulation efficiencies, which was explained by the unique mixing patterns achieved within the device. The microfluidic devices developed here demonstrate high potential as platforms for the discovery of novel MAPs as well as for other applications in the biomedical field such as the encapsulation and controlled delivery of bioactive compounds.