Binding affinity-based intracellular drug detection enabled by a unimolecular cucurbit[7]uril-dye conjugate.

Label-free fluorescence-based chemosensing has been increasingly brought into focus due to its simplicity and high sensitivity for intracellular monitoring of molecules. Currently used methods, such as conventional indicator displacement assays (IDAs), pose limitations related to dissociation upon dilution, random diffusion of the released indicators, and high sensitivity to interference by agents from the ambient cellular environment (e.g., salts, enzymes, and proteins). Herein we report a potentially widely applicable strategy to overcome the limitations of conventional IDAs by employing a macrocyclic cucurbit[7]uril (CB7) host covalently coupled to a nitrobenzoxadiazole (NBD) fluorescent dye (CB7-NBD conjugate). As a proof of concept, we demonstrated that the CB7-NBD unimolecular conjugate responded to various target analytes even in the complex live cell system. Moreover, the sensing system was compatible with fluorescence imaging, fluorescence-assisted cell sorting (FACS), and fluorescence spectrometry with a microplate reader. These experiments demonstrated an application of covalently bound unimolecular CB7-NBD conjugate as a sensor for detecting diverse analytes in the intracellular compartment of live cells.

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.

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.

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.

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.

Preparation and Characterization of an Injectable and Photo-Responsive Chitosan Methacrylate/Graphene Oxide Hydrogel: Potential Applications in Bone Tissue Adhesion and Repair.

As life expectancy continues to increase, the inevitable weakening and rupture of bone tissue have grown as concerns in the medical community, thus leading to the need for adhesive materials suitable for bone repair applications. However, current commercially available adhesives face certain drawbacks that prevent proper tissue repair, such as low biocompatibility, poor adhesion to wet surfaces, and the need for high polymerization temperatures. This work aims to develop an injectable and photo-responsive chitosan methacrylate/graphene oxide (ChiMA/GO) adhesive nanocomposite hydrogel of high biocompatibility that is easy to apply by simple extrusion and that offers the possibility for in situ polymer and physiological temperatures. The nanocomposite was thoroughly characterized spectroscopically, microscopically, rheologically, thermally, and through mechanical, textural, and biological assays to fully evaluate its correct synthesis and functionalization and its performance under physiological conditions that mimic those observed in vivo. In addition, a finite element analysis (FEA) simulation was used to evaluate its performance in femur fractures. Results suggest the material's potential as a bioadhesive, as it can polymerize at room temperature, shows superior stability in physiological media, and is capable of withstanding loads from body weight and movement. Moreover, the material showed remarkable biocompatibility as evidenced by low hemolytic and intermediate platelet aggregation tendencies, and high cytocompatibility when in contact with osteoblasts. The comprehensive studies presented here strongly suggest that the developed hydrogels are promising alternatives to conventional bone adhesives that might be further tested in vivo in the near future.

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.

Formulation and Characterization of a SIS-Based Photocrosslinkable Bioink.

Decellularized extracellular matrices (dECMs) represent a promising alternative as a source of materials to develop scaffolds that closely mimic the native environment of cells. As a result, dECMs have attracted significant attention for their applications in regenerative medicine and tissue engineering. One such application is 3D bioprinting, in which dECMs can be used to prepare bioinks with the biomimicry attributes required for regeneration purposes. Formulating bioinks is, however, challenging, due to difficulties in assuring that the printed materials match the mechanical properties of the tissue which is to be regenerated. To tackle this issue, a number of strategies have been devised, including crosslinking methods, the addition of synthetic materials as excipients, and the use of synthetic matrices for casting. We are particularly interested in extrusion-based 3D bioprinting, mainly due to the ease of rapidly conducting tests for adjusting operating conditions such that the required rheological and mechanical properties are met when using it. Here, we propose a novel bioink that consists of an acid-based precipitation of a small intestinal submucosa (SIS) dECM. The formulated bioink also relies on photocrosslinking reactions to attempt to control gelation and ultimately the mechanical properties of the extruded material. Photoinitiation was explored with the aid of varying concentrations of riboflavin (RF). Manual extrusion and rheological flow tests confirmed the printability and shear-thinning behavior of all formulations. Photocrosslinking reactions, however, failed to promote a substantial increase in gelation, which was attributed to considerable entanglement of undigested collagen molecules. As a result, pendant amine groups thought to be involved in the photo-mediated reactions remain largely inaccessible. In silico computational fluid dynamics (CFD) simulations were implemented to determine shear stress values on the bioink along the exit of the printing nozzle. Moreover, we calculated a stability parameter as a means to estimate changes in the bioink stability during the extrusion process. Future studies should be directed toward assessing the role of temperature-induced gelation in the rheological properties of the bioink and the development of strategies to improve the efficiency of photocrosslinking processes.

Novel BUF2-magnetite nanobioconjugates with cell-penetrating abilities.

INTRODUCTION: One of the major challenges of modern pharmacology is the development of systems for the delivery of therapeutic molecules in a controlled and localized manner. One strategy is to use nanostructured supports, which are well suited to carry a large number of molecules on a per mass basis. A major challenge for these supports is, however, their limited ability to bypass the cell membrane. Recent studies propose that to overcome this issue, potent translocating cell-penetrating peptides (CPPs) can be conjugated to their surfaces. METHODS: Here, we conjugated the antimicrobial CPP buforin II (BUF2) to the surface of magnetite nanoparticles to enhance their cell penetration. Conjugates were characterized via Fourier transform infrared spectroscopy, dynamic light scattering, and thermogravimetric analysis, and their biocompatibility was corroborated. The conjugates were delivered in both bacterial and mammalian cells demonstrating the intracellular inclusion in THP-1 cells for the first time. RESULTS: Despite the promising outcome, our studies showed that the obtained conjugates failed to maintain the native antimicrobial activity of BUF2. We hypothesize that to overcome this issue, a flexible linker can be inserted prior to conjugation. CONCLUSION: Our study highlights the potential of BUF2-magnetite conjugates as cell-penetrating vehicles for the targeted delivery of pharmacological agents. This provides support for the idea of a promising combined drug delivery and antimicrobial peptide therapy.