Effect of Exogenous pH on Cell Growth of Breast Cancer Cells.

Breast cancer is the most common type of cancer in women and the most life-threatening cancer in females worldwide. One key feature of cancer cells, including breast cancer cells, is a reversed pH gradient which causes the extracellular pH of cancer cells to be more acidic than that of normal cells. Growing literature suggests that alkaline therapy could reverse the pH gradient back to normal and treat the cancer; however, evidence remains inconclusive. In this study, we investigated how different exogenous pH levels affected the growth, survival, intracellular reactive oxygen species (ROS) levels and cell cycle of triple-negative breast cancer cells from MDA-MB-231 cancer cell lines. Our results demonstrated that extreme acidic conditions (pH 6.0) and moderate to extreme basic conditions (pH 8.4 and pH 9.2) retarded cellular growth, induced cell death via necrosis and apoptosis, increased ROS levels, and shifted the cell cycle away from the G0/G1 phase. However, slightly acidic conditions (pH 6.7) increased cellular growth, decreased ROS levels, did not cause significant cell death and shifted the cell cycle from the G0/G1 phase to the G2/M phase, thereby explaining why cancer cells favored acidic conditions over neutral ones. Interestingly, our results also showed that cellular pH history did not significantly affect the subsequent growth of cells when the pH of the medium was changed. Based on these results, we suggest that controlling or maintaining an unfavorable pH (such as a slightly alkaline pH) for cancer cells in vivo could retard the growth of cancer cells or potentially treat the cancer.

Inhibition of the Early-Stage Cross-Amyloid Aggregation of Amyloid-ß and IAPP via EGCG: Insights from Molecular Dynamics Simulations.

Amyloid-ß (Aß) and islet amyloid polypeptide (IAPP) are small peptides that have the potential to not only self-assemble but also cross-assemble and form cytotoxic amyloid aggregates. Recently, we experimentally investigated the nature of Aß-IAPP coaggregation and its inhibition by small polyphenolic molecules. Notably, we found that epigallocatechin gallate (EGCG) had the ability to reduce heteroaggregate formation. However, the precise molecular mechanism behind the reduction of heteroaggregates remains unclear. In this study, the dimerization processes of Aß40 and IAPP peptides with and without EGCG were characterized by the enhanced sampling technique. Our results showed that these amyloid peptides exhibited a tendency to form a stable heterodimer, which represented the first step toward coaggregation. Furthermore, we also found that the EGCG regulated the dimerization process. In the presence of EGCG, well-tempered metadynamics simulation indicated a notable shift in the bound state toward a greater center of mass (COM) distance. Additionally, the presence of EGCG led to a significant increase in the free energy barrier height (∼15k B T) along the COM distance, and we observed a transition state between the bound and unbound states. Our findings also unveiled that the EGCG formed a greater number of hydrogen bonds with Aß40, effectively obstructing the dimer formation. In addition, we carried out microseconds of all-atom conventional molecular dynamics (cMD) simulations to investigate the formation of both hetero- and homo-oligomer states by these peptides. MD simulations illustrated that EGCG played a significant role in preventing oligomer formation by reducing the content of ß-sheets in the peptide. Collectively, our results offered valuable insight into the mechanism of cross-amyloid aggregation between Aß40 and IAPP and the inhibition effect of EGCG on the heteroaggregation process.

3D-Printed Microcubes for Catalase Drug Delivery.

Oxidative stress, i.e., excessive production of reactive oxygen species (ROS), plays an important role in the pathogenesis of inflammatory diseases such as cardiovascular diseases, cancer, and neurodegenerative diseases. Catalase, an antioxidant enzyme, has great therapeutic potential; however, its efficacy is limited by its delivery to target cells or tissues. In order to achieve efficient delivery, consistent drug distribution, and drug activity, small and uniformly sized drug delivery vehicles are needed. Here, three-dimensional (3D) microcubes were printed by Nanoscribe Photonic Professional GT2, a high-resolution 3D printer, and the characteristics of 3D-printed microcubes as drug delivery vehicles for the delivery of catalase were investigated. The size of the 3D-printed microcubes was 800 nm in length of a square and 600 nm in height, which is suitable for targeting macrophages passively. Microcubes were also tunable in shape and size, and high-resolution 3D printing could provide microparticles with little variation in shape and size. Catalase was loaded on 3D-printed microcubes by nonspecific adsorption, and catalase on 3D-printed microcubes (CAT-MC) retained 83.1 ± 1.3% activity of intact catalase. CAT-MC also saved macrophages, RAW 264.7, from the cytotoxicity of H2O2 by 86.4 ± 4.1%. As drug delivery vehicles, 3D-printed microparticles are very promising due to their small and uniform size, which provides consistent drug distribution and drug activity. Therefore, we anticipate numerous applications of 3D-printed microparticles for delivering therapeutic proteins.

2D MXenes for controlled releases of therapeutic proteins.

MXenes belong to a new class of two dimensional (2D) functional nanomaterials, mainly encompassing transition-metal carbides, nitrides and carbonitrides, with unique physical, chemical, electronic and mechanical properties for various emerging applications across different fields. To date, the potentials of MXenes for biomedical application such as drug delivery have not been thoroughly explored due to the lack of information on their biocompatibility, cytotoxicity and biomolecule-surface interaction. In this study, we developed novel drug delivery system from MXene for the controlled release of a model therapeutic protein. First, the structural, chemical and morphological properties of as synthesized MXenes were probed with electron microscopy and X-ray diffraction. Second, the potential cytotoxicity of MXene toward the proliferation and cell morphology of murine macrophages (RAW 264.7) were evaluated with MTT assays and electron microscopy, respectively. Moreover, the drug loading capacities and sustained release capabilities of MXene were assessed in conjunction with machine learning approaches. Our results demonstrated that MXene did not significantly induce cellular toxicity at any concentration below 1 mg/ml which is within the range for effective dose of drug delivery vehicle. Most importantly, MXene was efficiently loaded with FITC-catalase for subsequently achieving controlled release under different pHs. The release profiles of catalase from MXene showed higher initial rate under basic buffer (pH 9) compared to that in physiological (pH 7.4) and acidic buffers (pH 2). Taken together, the results of this study lead to a fundamental advancement toward the use of MXene as a nanocarrier for therapeutic proteins in drug delivery applications.

Hydrogen phosphate selectively induces MDA MB 231 triple negative breast cancer cell death in vitro.

Phosphate ions are the most abundant anions inside the cells, and they are increasingly gaining attention as key modulators of cellular function and gene expression. However, little is known about the effect of inorganic phosphate ions on cancer cells, particularly breast cancer cells. Here, we investigated the toxicity of different phosphate compounds to triple-negative human breast cancer cells, particularly, MDA-MB-231, and compared it to that of human monocytes, THP-1. We found that, unlike dihydrogen phosphate (H2PO4-), hydrogen phosphate (HPO42-) at 20 mM or lower concentrations induced breast cancer cell death more than immune cell death, mainly via apoptosis. We correlate this effect to the fact that phosphate in the form of HPO42- raises pH levels to alkaline levels which are not optimum for transport of phosphate into cancer cells. The results in this study highlight the importance of further exploring hydrogen phosphate (HPO42-) as a potential therapeutic for the treatment of breast cancer.

Linking Alzheimer's Disease and Type 2 Diabetes: Characterization and Inhibition of Cytotoxic Aß and IAPP Hetero-Aggregates.

The cytotoxic self-aggregation of ß-amyloid (Aß) peptide and islet amyloid polypeptide (IAPP) is implicated in the pathogenesis of Alzheimer's disease (AD) and Type 2 diabetes (T2D), respectively. Increasing evidence, particularly the co-deposition of Aß and IAPP in both brain and pancreatic tissues, suggests that Aß and IAPP cross-interaction may be responsible for a pathological link between AD and T2D. Here, we examined the nature of IAPP-Aß40 co-aggregation and its inhibition by small molecules. In specific, we characterized the kinetic profiles, morphologies, secondary structures and toxicities of IAPP-Aß40 hetero-assemblies and compared them to those formed by their homo-assemblies. We demonstrated that monomeric IAPP and Aß40 form stable hetero-dimers and hetero-assemblies that further aggregate into ß-sheet-rich hetero-aggregates that are toxic (cell viability <50%) to both PC-12 cells, a neuronal cell model, and RIN-m5F cells, a pancreatic cell model for ß-cells. We then selected polyphenolic candidates to inhibit IAPP or Aß40 self-aggregation and examined the inhibitory effect of the most potent candidate on IAPP-Aß40 co-aggregation. We demonstrated that epigallocatechin gallate (EGCG) form inter-molecular hydrogen bonds with each of IAPP and Aß40. We also showed that EGCG reduced hetero-aggregate formation and resulted in lower ß-sheets content and higher unordered structures in IAPP-Aß40-EGCG samples. Importantly, we showed that EGCG is highly effective in reducing the toxicity of IAPP-Aß40 hetero-aggregates on both cell models, specifically at concentrations that are equivalent to or are 2.5-fold higher than the mixed peptide concentrations. To the best of our knowledge, this is the first study to report the inhibition of IAPP-Aß40 co-aggregation by small molecules. We conclude that EGCG is a promising candidate to prevent co-aggregation and cytotoxicity of IAPP-Aß40, which in turn, contribute to the pathological link between AD and T2D.

Lymph Nodes-On-Chip: Promising Immune Platforms for Pharmacological and Toxicological Applications.

Organs-on-chip are gaining increasing attention as promising platforms for drug screening and testing applications. However, lymph nodes-on-chip options remain limited although the lymph node is one of the main determinants of the immunotoxicity of newly developed pharmacological drugs. In this review, we describe existing biomimetic lymph nodes-on-chip, their design, and their physiological relevance to pharmacology and shed the light on future directions associated with lymph node-on-chip design and implementation in drug discovery and development.

Nanocomposite Conductive Bioinks Based on Low-Concentration GelMA and MXene Nanosheets/Gold Nanoparticles Providing Enhanced Printability of Functional Skeletal Muscle Tissues.

There is a growing need to develop novel well-characterized biological inks (bioinks) that are customizable for three-dimensional (3D) bioprinting of specific tissue types. Gelatin methacryloyl (GelMA) is one such candidate bioink due to its biocompatibility and tunable mechanical properties. Currently, only low-concentration GelMA hydrogels (≤5% w/v) are suitable as cell-laden bioinks, allowing high cell viability, elongation, and migration. Yet, they offer poor printability. Herein, we optimize GelMA bioinks in terms of concentration and cross-linking time for improved skeletal muscle C2C12 cell spreading in 3D, and we augment these by adding gold nanoparticles (AuNPs) or a two-dimensional (2D) transition metal carbide (MXene nanosheets) for enhanced printability and biological properties. AuNP and MXene addition endowed GelMA with increased conductivity (up to 0.8 ± 0.07 and 0.9 ± 0.12 S/m, respectively, compared to 0.3 ± 0.06 S/m for pure GelMA). Furthermore, it resulted in an improvement of rheological properties and printability, specifically at 10 °C. Improvements in electrical and rheological properties led to enhanced differentiation of encapsulated myoblasts and allowed for printing highly viable (97%) stable constructs. Taken together, these results constitute a significant step toward fabrication of 3D conductive tissue constructs with physiological relevance.

Multi-Compartment Lymph-Node-on-a-Chip Enables Measurement of Immune Cell Motility in Response to Drugs.

Organs On-a-Chip represent novel platforms for modelling human physiology and disease. The lymph node (LN) is a relevant immune organ in which B and T lymphocytes are spatially organized in a complex architecture, and it is the place where the immune response initiates. The present study addresses the utility of a recently designed LN-on-a-chip to dissect and understand the effect of drugs delivered to cells in a fluidic multicellular 3D setting that mimics the human LN. To do so, we analyzed the motility and viability of human B and T cells exposed to hydroxychloroquine (HCQ). We show that the innovative LN platform, which operates at a microscale level, allows real-time monitoring of co-cultured B and T cells by imaging, and supports cellular random movement. HCQ delivered to cells through a constant and continuous flow induces a reduction in T cell velocity while promotes persistent rotational motion. We also find that HCQ increases the production of reactive oxygen species in T cells. Taken together, these results highlight the potential of the LN-on-a-chip to be applied in drug screening and development, and in cellular dynamics studies.

Inhibition of SARS-CoV-2 Entry into Host Cells Using Small Molecules.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a virus belonging to the Coronavirus family, is now known to cause Coronavirus Disease (Covid-19) which was first recognized in December 2019. Covid-19 leads to respiratory illnesses ranging from mild infections to pneumonia and lung failure. Strikingly, within a few months of its first report, Covid-19 has spread worldwide at an exceptionally high speed and it has caused enormous human casualties. As yet, there is no specific treatment for Covid-19. Designing inhibitory drugs that can interfere with the viral entry process constitutes one of the main preventative therapies that could combat SARS-CoV-2 infection at an early stage. In this review, we provide a brief introduction of the main features of coronaviruses, discuss the entering mechanism of SARS-CoV-2 into human host cells and review small molecules that inhibit SARS-CoV-2 entry into host cells. Specifically, we focus on small molecules, identified by experimental validation and/or computational prediction, that target the SARS-CoV-2 spike protein, human angiotensin converting enzyme 2 (ACE2) receptor and the different host cell proteases that activate viral fusion. Given the persistent rise in Covid-19 cases to date, efforts should be directed towards validating the therapeutic effectiveness of these identified small molecule inhibitors.

Multi-Compartment 3D-Cultured Organ-on-a-Chip: Towards a Biomimetic Lymph Node for Drug Development.

The interaction of immune cells with drugs and/or with other cell types should be mechanistically investigated in order to reduce attrition of new drug development. However, they are currently only limited technologies that address this need. In our work, we developed initial but significant building blocks that enable such immune-drug studies. We developed a novel microfluidic platform replicating the Lymph Node (LN) microenvironment called LN-on-a-chip, starting from design all the way to microfabrication, characterization and validation in terms of architectural features, fluidics, cytocompatibility, and usability. To prove the biomimetics of this microenvironment, we inserted different immune cell types in a microfluidic device, which showed an in-vivo-like spatial distribution. We demonstrated that the developed LN-on-a-chip incorporates key features of the native human LN, namely, (i) similarity in extracellular matrix composition, morphology, porosity, stiffness, and permeability, (ii) compartmentalization of immune cells within distinct structural domains, (iii) replication of the lymphatic fluid flow pattern, (iv) viability of encapsulated cells in collagen over the typical timeframe of immunotoxicity experiments, and (v) interaction among different cell types across chamber boundaries. Further studies with this platform may assess the immune cell function as a step forward to disclose the effects of pharmaceutics to downstream immunology in more physiologically relevant microenvironments.

In Vitro Immune Organs-on-Chip for Drug Development: A Review.

The current drug development practice lacks reliable and sensitive techniques to evaluate the immunotoxicity of drug candidates, i.e., their effect on the human immune system. This, in part, has resulted in a high attrition rate for novel drugs candidates. Organ-on-chip devices have emerged as key tools that permit the study of human physiology in controlled in vivo simulating environments. Furthermore, there has been a growing interest in developing the so called "body-on-chip" devices to better predict the systemic effects of drug candidates. This review describes existing biomimetic immune organs-on-chip, highlights their physiological relevance to drug development and discovery and emphasizes the need for developing comprehensive immune system-on-chip models. Such immune models can enhance the performance of novel drug candidates during clinical trials and contribute to reducing the high attrition rate as well as the high cost associated with drug development.

A spatiotemporal characterization method for the dynamic cytoskeleton.

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (Df ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to Df , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in Df and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 Wiley Periodicals, Inc.