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.

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.

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.

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.

Recent Advances on Stimuli-Responsive Hydrogels Based on Tissue-Derived ECMs and Their Components: Towards Improving Functionality for Tissue Engineering and Controlled Drug Delivery.

Due to their highly hydrophilic nature and compositional versatility, hydrogels have assumed a protagonic role in the development of physiologically relevant tissues for several biomedical applications, such as in vivo tissue replacement or regeneration and in vitro disease modeling. By forming interconnected polymeric networks, hydrogels can be loaded with therapeutic agents, small molecules, or cells to deliver them locally to specific tissues or act as scaffolds for hosting cellular development. Hydrogels derived from decellularized extracellular matrices (dECMs), in particular, have gained significant attention in the fields of tissue engineering and regenerative medicine due to their inherently high biomimetic capabilities and endowment of a wide variety of bioactive cues capable of directing cellular behavior. However, these hydrogels often exhibit poor mechanical stability, and their biological properties alone are not enough to direct the development of tissue constructs with functional phenotypes. This review highlights the different ways in which external stimuli (e.g., light, thermal, mechanical, electric, magnetic, and acoustic) have been employed to improve the performance of dECM-based hydrogels for tissue engineering and regenerative medicine applications. Specifically, we outline how these stimuli have been implemented to improve their mechanical stability, tune their microarchitectural characteristics, facilitate tissue morphogenesis and enable precise control of drug release profiles. The strategic coupling of the bioactive features of dECM-based hydrogels with these stimulation schemes grants considerable advances in the development of functional hydrogels for a wide variety of applications within these fields.

Graphene Oxide-Embedded Extracellular Matrix-Derived Hydrogel as a Multiresponsive Platform for 3D Bioprinting Applications.

Decellularized extracellular matrices (dECMs) have shown enormous potential for the biofabrication of tissues due to their biomimetic properties that promote enhanced cellular interaction and tissue regeneration. However, biofabrication schemes requiring electrostimulation pose an additional constraint due to the insulating properties of natural materials. Here, we propose a methacryloyl-modified decellularized small intestine submucosa (SISMA) hydrogel, embedded with graphene oxide (GO) nanosheets, for extrusion-based 3D bioprinting applications that require electrostimulation. Methacryloyl biochemical modification is performed to enhance the mechanical stability of dECM constructs by mediating photo-crosslinking reactions, and a multistep fabrication scheme is proposed to harness the bioactive and hydrophilic properties of GO and electroconductive properties of reduced GO. For this, GO was initially dispersed in SISMA hydrogels by exploiting its hydrophilicity and protein adsorption capabilities, and in situ reduction was subsequently performed to confer electroconductive abilities. SISMA-GO composite hydrogels were successfully prepared with enhanced structural characteristics, as shown by the higher crosslinking degree and increased elastic response upon blue-light exposure. Moreover, GO was homogeneously dispersed without affecting photocrosslinking reactions and hydrogel shear-thinning properties. Human adipose-derived mesenchymal stem cells were successfully bioprinted in SISMA-GO with high cell viability after 1 week and in situ reduction of GO during this period enhanced the electrical conductivity of these nanostructures. This work demonstrates the potential of SISMA-GO bioinks as bioactive and electroconductive scaffolds for electrostimulation applications in tissue engineering and regenerative medicine.

Tailoring Iron Oxide Nanoparticles for Efficient Cellular Internalization and Endosomal Escape.

Iron oxide nanoparticles (IONs) have been widely explored for biomedical applications due to their high biocompatibility, surface-coating versatility, and superparamagnetic properties. Upon exposure to an external magnetic field, IONs can be precisely directed to a region of interest and serve as exceptional delivery vehicles and cellular markers. However, the design of nanocarriers that achieve an efficient endocytic uptake, escape lysosomal degradation, and perform precise intracellular functions is still a challenge for their application in translational medicine. This review highlights several aspects that mediate the activation of the endosomal pathways, as well as the different properties that govern endosomal escape and nuclear transfection of magnetic IONs. In particular, we review a variety of ION surface modification alternatives that have emerged for facilitating their endocytic uptake and their timely escape from endosomes, with special emphasis on how these can be manipulated for the rational design of cell-penetrating vehicles. Moreover, additional modifications for enhancing nuclear transfection are also included in the design of therapeutic vehicles that must overcome this barrier. Understanding these mechanisms opens new perspectives in the strategic development of vehicles for cell tracking, cell imaging and the targeted intracellular delivery of drugs and gene therapy sequences and vectors.