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Abstract 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. more...

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. more...

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. more...

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. more...

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. more...

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. more...

The discovery and development of novel pharmaceuticals is an area of active research mainly due to the large investments required and long payback times. As of 2016, the development of a novel drug candidate required up to $ USD 2.6 billion in investment for only 10% rate of approval by the FDA. To help decreasing the costs associated with the process, a number of in silico approaches have been developed with relatively low success due to limited predicting performance. Here, we introduced a machine learning-based algorithm as an alternative for a more accurate search of new pharmacological candidates, which takes advantage of Recurrent Neural Networks (RNN) for active molecule prediction within large databases. Our approach, termed PharmaNet was implemented here to search for ligands against specific cell receptors within 102 targets of the DUD-E database, which contains 22886 active molecules. PharmaNet comprises three main phases. First, a SMILES representation of the molecule is converted into a raw molecular image. Second, a convolutional encoder processes the data to obtain a fingerprint molecular image that is finally analyzed by a Recurrent Neural Network (RNN). This approach enables precise predictions of the molecules' target on the basis of the feature extraction, the sequence analysis and the relevant information filtered out throughout the process. Molecule Target prediction is a highly unbalanced detection problem and therefore, we propose that an adequate evaluation metric of performance is the area under the Normalized Average Precision (NAP) curve. PharmaNet largely surpasses the previous state-of-the-art method with 97.7% in the Receiver Operating Characteristic curve (ROC-AUC) and 65.5% in the NAP curve. We obtained a perfect performance for human farnesyl pyrophosphate synthase (FPPS), which is a potential target for antimicrobial and anticancer treatments. We decided to test PharmaNet for activity prediction against FPPS by searching in the CHEMBL data set. We obtained three (3) potential inhibitors that were further validated through both molecular docking and in silico toxicity prediction. Most importantly, one of this candidates, CHEMBL2007613, was predicted as a potential antiviral due to its involvement on the PCDH17 pathway, which has been reported to be related to viral infections. more...

At the beginning of 2020, the pandemic caused by the SARS-CoV-2 virus led to the fast sequencing of its genome to facilitate molecular engineering strategies to control the pathogen’s spread. The spike (S) glycoprotein has been identified as the leading therapeutic agent due to its role in localizing the ACE2 receptor in the host’s pulmonary cell membrane, binding, and eventually infecting the cells. Due to the difficulty of delivering bioactive molecules to the intracellular space, we hypothesized that the S protein could serve as a source of membrane translocating peptides. AHB-1, AHB-2, and AHB-3 peptides were identified and analyzed on a membrane model of DPPC (dipalmitoylphosphatidylcholine) using molecular dynamics (MD) simulations. An umbrella sampling approach was used to quantify the energy barrier necessary to cross the boundary (13.2 to 34.9 kcal/mol), and a flat-bottom pulling helped to gain a deeper understanding of the membrane’s permeation dynamics. Our studies revealed that the novel peptide AHB-1 exhibited comparable penetration potential of already known potent cell-penetrating peptides (CPPs) such as TP2, Buforin II, and Frenatin 2.3s. Results were confirmed by in vitro analysis of the peptides conjugated to chitosan nanoparticles, demonstrating its ability to reach the cytosol and escape endosomes, while maintaining high biocompatibility levels according to standardized assays. more...

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. more...

One of the challenges of modern biotechnology is to find new routes to mitigate the resistance to conventional antibiotics. Antimicrobial peptides (AMPs) are an alternative type of biomolecules, naturally present in a wide variety of organisms, with the capacity to overcome the current microorganism resistance threat. Here, we reviewed our recent efforts to develop a new library of non-rationally produced AMPs that relies on bacterial genome inherent diversity and compared it with rationally designed libraries. Our approach is based on a four-stage workflow process that incorporates the interplay of recent developments in four major emerging technologies: artificial intelligence, molecular dynamics, surface-display in microorganisms, and microfluidics. Implementing this framework is challenging because to obtain reliable results, the in silico algorithms to search for candidate AMPs need to overcome issues of the state-of-the-art approaches that limit the possibilities for multi-space data distribution analyses in extremely large databases. We expect to tackle this challenge by using a recently developed classification algorithm based on deep learning models that rely on convolutional layers and gated recurrent units. This will be complemented by carefully tailored molecular dynamics simulations to elucidate specific interactions with lipid bilayers. Candidate AMPs will be recombinantly-expressed on the surface of microorganisms for further screening via different droplet-based microfluidic-based strategies to identify AMPs with the desired lytic abilities. We believe that the proposed approach opens opportunities for searching and screening bioactive peptides for other applications. more...

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. more...

Emerging water pollutants such as pharmaceutical contaminants are suspected to induce adverse effects to human health. These molecules became worrisome due to their increasingly high concentrations in surface waters. Despite this alarming situation, available data about actual concentrations in the environment is rather scarce, as it is not commonly monitored or regulated. This is aggravated even further by the absence of portable and reliable methods for their determination in the field. A promising way to tackle these issues is the use of enzyme-based and miniaturized biosensors for their electrochemical detection. Here, we present an overview of the latest developments in amperometric microfluidic biosensors that include, modeling and multiphysics simulation, design, manufacture, testing, and operation methods. Different types of biosensors are described, highlighting those based on oxidases/peroxidases and the integration with microfluidic platforms. Finally, issues regarding the stability of the biosensors and the enzyme molecules are discussed, as well as the most relevant approaches to address these obstacles. more...