Investigation of the ionic conditions in SiRNA-mediated delivery through its carriers in the cell membrane: a molecular dynamic simulation.

SiRNA is a new generation of drug molecules and a new approach for treating a variety of diseases such as cancer and viral infections. SiRNA delivery to cells and translocation into cytoplasm are the main challenges in the clinical application of siRNA. Lipid carriers are one of the most successful carriers for siRNA delivery. In this study, we investigated the interaction of siRNA with a zwitterionic bilayer and how ion concentration and lipid conjugation can affect it. The divalent cation such as Mg2+ ions could promote the siRNA adsorption on the bilayer surface. The cation ions can bind to the head groups of lipids and the grooves of siRNA molecules and form bridges between the siRNA and bilayer surface. Our findings demonstrated the bridges formed by divalent ions could facilitate the attachment of siRNA to the membrane surface. We showed that the divalent cations can regulate the bridging-driven membrane attachment and it seems the result of this modulation can be used for designing biomimetic devices. In the following, we examined the effect of cations on the interaction between siRNA modified by cholesterol and the membrane surface. Our MD simulations showed that in the presence of Mg2+, the electrostatic and vdW energy between the membrane and siRNA were higher compared to those in the presence of NA+. We showed that the electrostatic interaction between membrane and siRNA cannot be facilitated only by cholesterol conjugated. Indeed, cations are essential to create coulomb repulsion and enable membrane attachment. This study provides important insight into liposome carriers for siRNA delivery and could help us in the development of siRNA-based therapeutics. Due to the coronavirus pandemic outbreak, these results may shed light on the new approach for treating these diseases and their molecular details.

Differentiation of PC12 cell line into neuron by Valproic acid encapsulated in the stabilized core-shell liposome-chitosan Nano carriers.

Valproic acid (VPA) usage in high dose is teratogen with low bioavailability. Hence to improve its efficacy and reduce its side effect it was encapsulated by the Nano liposomes and stabilized by the chitosan at different concentrations. The cellular uptake, biocompatibility, loading and encapsulation efficiency of the six-different formulations (1:1, 2:1, and 4:1 of chitosan-phospholipids: VPA), PC12 differentiation to neuron cells assays (gene-expression level by qRT-PCR) were conducted for the efficacy assessment of the Nano carriers. The encapsulation efficiency (EE) results revealed that the encapsulation of the VPA corresponds to the phospholipids dose, where 2:1 formulations showed higher encapsulating rate (64.5% for non-coated and 80% for coated by chitosan). The time monitored released of VPA also showed that the chitosan could enhance its controlled release too. The cellular uptake exhibited similar uptake behavior for both the coated and the non-coated Nano carriers and cytoplasmic distribution. We witnessed no toxicity effects, at different concentrations, for both formulations. Moreover, the results indicated that the gene expression level of SOX2, NeuroD1, and Neurofilament 200 increased from 1 to 5 folds for different genes. The qRT-PCR data were confirmed by the immunofluorescence antibodies staining, where Neurofilament 68 and SOX2 cell markers were modulated during differentiation of PC12 cells. Finally, our findings suggest promising potential for the Lip-VPA-Chit Nano carrier in inducing the differentiation of PC12 into neuron for treating neurodegenerative disorders.

Fingerprinting Metabolic Activity and Tissue Integrity of 3D Lung Cancer Spheroids under Gold Nanowire Treatment.

Inadequacy of most animal models for drug efficacy assessments has led to the development of improved in vitro models capable of mimicking in vivo exposure scenarios. Among others, 3D multicellular spheroid technology is considered to be one of the promising alternatives in the pharmaceutical drug discovery process. In addition to its physiological relevance, this method fulfills high-throughput and low-cost requirements for preclinical cell-based assays. Despite the increasing applications of spheroid technology in pharmaceutical screening, its application, in nanotoxicity testing is still in its infancy due to the limited penetration and uptake rates into 3D-cell assemblies. To gain a better understanding of gold nanowires (AuNWs) interactions with 3D spheroids, a comparative study of 2D monolayer cultures and 3D multicellular spheroids was conducted using two lung cancer cell lines (A549 and PC9). Cell apoptosis (live/dead assay), metabolic activity, and spheroid integrity were evaluated following exposure to AuNWs at different dose-time manners. Results revealed a distinct different cellular response between 2D and 3D cell cultures during AuNWs treatment including metabolic rates, cell viability, dose-response curves and, uptake rates. Our data also highlighted further need for more physiologically relevant tissue models to investigate in depth nanomaterial-biology interactions. It is important to note that higher concentrations of AuNWs with lower exposure times and lower concentrations of AuNWs with higher exposure times of 3 days resulted in the loss of spheroid integrity by disrupting cell-cell contacts. These findings could help to increase the understanding of AuNWs-induced toxicity on tissue levels and also contribute to the establishment of new analytical approaches for toxicological and drug screening studies.

Microfluidic investigation of the effect of graphene oxide on mechanical properties of cell and actin cytoskeleton networks: experimental and theoretical approaches.

Biomechanical and morphological analysis of the cells is a novel approach for monitoring the environmental features, drugs, and toxic compounds' effects on cells. Graphene oxide (GO) has a broad range of medical applications such as tissue engineering and drug delivery. However, the effects of GO nanosheets on biological systems have not been completely understood. In this study, we focused on the biophysical characteristics of cells and their changes resulting from the effect of GO nanosheets. The biophysical properties of the cell population were characterized as follows: cell stiffness was calculated by atomic force microscopy, cell motility and invasive properties were characterized in the microfluidic chip in which the cells are able to visualize cell migration at a single-cell level. Intracellular actin was stained to establish a quantitative picture of the intracellular cytoskeleton. In addition, to understand the molecular interaction of GO nanosheets and actin filaments, coarse-grained (CG) molecular dynamics (MD) simulations were carried out. Our results showed that GO nanosheets can reduce cell stiffness in MCF7 cells and MDA-MB-231 cell lines and highly inhibited cell migration (39.2%) in MCF-7 and (38.6%) in MDA-MB-231 cell lines through the GO nanosheets-mediated disruption of the intracellular cytoskeleton. In the presence of GO nanosheets, the cell migration of both cell lines, as well as the cell stiffness, significantly decreased. Moreover, after GO nanosheets treatment, the cell actin network dramatically changed. The experimental and theoretical approaches established a quantitative picture of changes in these networks. Our results showed the reduction of the order parameter in actin filaments was 23% in the MCF7 cell line and 20.4% in the MDA-MB-231 cell line. The theoretical studies also showed that the GO nanosheet-actin filaments have stable interaction during MD simulation. Moreover, the 2D free energy plot indicated the GO nanosheet can induce conformational changes in actin filaments. Our findings showed that the GO nanosheets can increase the distance of actin-actin subunits from 3.22 to 3.5 nm and in addition disrupt native contacts between two subunits which lead to separate actin subunits from each other in actin filaments. In this study, the biomechanical characteristics were used to explain the effect of GO nanosheets on cells which presents a novel view of how GO nanosheets can affect the biological properties of cells without cell death. These findings have the potential to be applied in different biomedical applications.

A combined microfluidic deep learning approach for lung cancer cell high throughput screening toward automatic cancer screening applications.

Lung cancer is a leading cause of cancer death in both men and women worldwide. The high mortality rate in lung cancer is in part due to late-stage diagnostics as well as spread of cancer-cells to organs and tissues by metastasis. Automated lung cancer detection and its sub-types classification from cell's images play a crucial role toward an early-stage cancer prognosis and more individualized therapy. The rapid development of machine learning techniques, especially deep learning algorithms, has attracted much interest in its application to medical image problems. In this study, to develop a reliable Computer-Aided Diagnosis (CAD) system for accurately distinguishing between cancer and healthy cells, we grew popular Non-Small Lung Cancer lines in a microfluidic chip followed by staining with Phalloidin and images were obtained by using an IX-81 inverted Olympus fluorescence microscope. We designed and tested a deep learning image analysis workflow for classification of lung cancer cell-line images into six classes, including five different cancer cell-lines (P-C9, SK-LU-1, H-1975, A-427, and A-549) and normal cell-line (16-HBE). Our results demonstrate that ResNet18, a residual learning convolutional neural network, is an efficient and promising method for lung cancer cell-lines categorization with a classification accuracy of 98.37% and F1-score of 97.29%. Our proposed workflow is also able to successfully distinguish normal versus cancerous cell-lines with a remarkable average accuracy of 99.77% and F1-score of 99.87%. The proposed CAD system completely eliminates the need for extensive user intervention, enabling the processing of large amounts of image data with robust and highly accurate results.

Preparation and Evaluation of Doxorubicin-Loaded PLA-PEG-FA Copolymer Containing Superparamagnetic Iron Oxide Nanoparticles (SPIONs) for Cancer Treatment: Combination Therapy with Hyperthermia and Chemotherapy.

BACKGROUND: Among the novel cancer treatment strategies, combination therapy is a cornerstone of cancer therapy. MATERIALS AND METHODS: Here, combination therapy with targeted polymer, magnetic hyperthermia and chemotherapy was presented as an effective therapeutic technique. The DOX-loaded PLA-PEG-FA magnetic nanoparticles (nanocarrier) were prepared via a double emulsion method. The nanocarriers were characterized by particle size, zeta potential, morphology, saturation magnetizations and heat generation capacity, and the encapsulation efficiency, drug content and in-vitro drug release for various weight ratios of PLA:DOX. Then, cytotoxicity, cellular uptake and apoptosis level of nanocarrier-treated cells for HeLa and CT26 cells were investigated by MTT assay, flow cytometry, and apoptosis detection kit. RESULTS AND CONCLUSIONS: The synthesized nanoparticles were spherical in shape, had low aggregation and considerable magnetic properties. Meanwhile, the drug content and encapsulation efficiency of nanoparticles can be achieved by varying the weight ratios of PLA:DOX. The saturation magnetizations of nanocarriers in the maximum applied magnetic field were 59/447 emu/g and 28/224 emu/g, respectively. Heat generation capacity of MNPs and nanocarriers were evaluated in the external AC magnetic field by a hyperthermia device. The highest temperature, 44.2°C, was measured in the nanocarriers suspension at w/w ratio 10:1 (polymer:DOX weight ratio) after exposed to the magnetic field for 60 minutes. The encapsulation efficiency improved with increasing polymer concentration, since the highest DOX encapsulation efficiency was related to the nanocarriers' suspension at w/w ratio 50:1 (79.6 ± 6.4%). However, the highest DOX loading efficiency was measured in the nanocarriers' suspension at w/w ratio 10:1 (5.14 ± 0.6%). The uptake efficiency and apoptosis level of nanocarrier-treated cells were higher than those of nanocarriers (folic acid free) and free DOX-treated cells in both cell lines. Therefore, this targeted nanocarrier may offer a promising nanosystem for cancer-combined chemotherapy and hyperthermia.

Biomedical Applications of TiO2 Nanostructures: Recent Advances.

Titanium dioxide (TiO2) nanostructures are one of the most plentiful compounds that have emerged in various fields of technology such as medicine, energy and biosensing. Various TiO2 nanostructures (nanotubes [NTs] and nanowires) have been employed in photoelectrochemical (PEC) biosensing applications, greatly enhancing the detection of targets. TiO2 nanostructures, used as reinforced material or coatings for the bare surface of titanium implants, are excellent additive materials to compensate titanium implants deficiencies-like poor surface interaction with surrounding tissues-by providing nanoporous surfaces and hierarchical structures. These nanostructures can also be loaded by diversified drugs-like osteoporosis drugs, anticancer and antibiotics-and used as local drug delivery systems. Furthermore, TiO2 nanostructures and their derivatives are new emerging antimicrobial agents to overcome human pathogenic microorganisms. However, like all other nanomaterials, toxicity and biocompatibility of TiO2 nanostructures must be considered. This review highlights recent advances, along with the properties and numerous applications of TiO2-based nanostructure compounds in nano biosensing, medical implants, drug delivery and antibacterial fields. Moreover, in the present study, some recent advances accomplished on the pharmaceutical applications of TiO2 nanostructures, as well as its toxicity and biocompatibility, are presented.

Capture and detection of rare cancer cells in blood by intrinsic fluorescence of a novel functionalized diatom.

The ability to identify and enrich target cells can play a significant role in biosensing in general. For the separation of rare cells; a biosilica structure was extracted from "Chaetoceros sp." diatoms as a novel natural source of mesoporous materials. These diatoms had special optical capabilities, especially in fluorescence emission. Biosilica surfaces of Chaetoceros sp. were chemically modified by iron oxide nanoparticles resulting in diatom silica magnetic particles functionalized with Trastuzumab antibody to separate the breast cancer cells from normal cells. The fully characterization of magnetic biosilica structure were studied by various spectroscopic techniques. The magnetic diatom conjugated with antibody displays strong absorption and two main types of fluorescence emission with peaks centered at 493 and 650 nm (photo-excited at 405 nm). As in vitro study, SKBR3 cells (HER2 positive cells) were selectively targeted and separated with this magnetic diatom structure from the mix of HER2 negative cells using a magnetic field. These results show that Chaetoceros silica shells are promising eco-friendly biomaterials suitable for biosensing chip and the targeted delivery of drugs to the specific sites.

PDMS Nano-Modified Scaffolds for Improvement of Stem Cells Proliferation and Differentiation in Microfluidic Platform.

Microfluidics cell-based assays require strong cell-substrate adhesion for cell viability, proliferation, and differentiation. The intrinsic properties of PDMS, a commonly used polymer in microfluidics systems, regarding cell-substrate interactions have limited its application for microfluidics cell-based assays. Various attempts by previous researchers, such as chemical modification, plasma-treatment, and protein-coating of PDMS revealed some improvements. These strategies are often reversible, time-consuming, short-lived with either cell aggregates formation, not cost-effective as well as not user- and eco-friendly too. To address these challenges, cell-surface interaction has been tuned by the modification of PDMS doped with different biocompatible nanomaterials. Gold nanowires (AuNWs), superparamagnetic iron oxide nanoparticles (SPIONs), graphene oxide sheets (GO), and graphene quantum dot (GQD) have already been coupled to PDMS as an alternative biomaterial enabling easy and straightforward integration during microfluidic fabrication. The synthesized nanoparticles were characterized by corresponding methods. Physical cues of the nanostructured substrates such as Young's modulus, surface roughness, and nanotopology have been carried out using atomic force microscopy (AFM). Initial biocompatibility assessment of the nanocomposites using human amniotic mesenchymal stem cells (hAMSCs) showed comparable cell viabilities among all nanostructured PDMS composites. Finally, osteogenic stem cell differentiation demonstrated an improved differentiation rate inside microfluidic devices. The results revealed that the presence of nanomaterials affected a 5- to 10-fold increase in surface roughness. In addition, the results showed enhancement of cell proliferation from 30% (pristine PDMS) to 85% (nano-modified scaffolds containing AuNWs and SPIONs), calcification from 60% (pristine PDMS) to 95% (PDMS/AuNWs), and cell surface marker expression from 40% in PDMS to 77% in SPION- and AuNWs-PDMS scaffolds at 14 day. Our results suggest that nanostructured composites have a very high potential for stem cell studies and future therapies.

Gold Nanowires/Fibrin Nanostructure as Microfluidics Platforms for Enhancing Stem Cell Differentiation: Bio-AFM Study.

Organ-on-a-chip technology has gained great interest in recent years given its ability to control the spatio-temporal microenvironments of cells and tissues precisely. While physical parameters of the respective niche such as microchannel network sizes, geometric features, flow rates, and shear forces, as well as oxygen tension and concentration gradients, have been optimized for stem cell cultures, little has been done to improve cell-matrix interactions in microphysiological systems. Specifically, detailed research on the effect of matrix elasticity and extracellular matrix (ECM) nanotopography on stem cell differentiation are still in its infancy, an aspect that is known to alter a stem cell's fate. Although a wide range of hydrogels such as gelatin, collagen, fibrin, and others are available for stem cell chip cultivations, only a limited number of elasticities are generally employed. Matrix elasticity and the corresponding nanotopography are key factors that guide stem cell differentiation. Given this, we investigated the addition of gold nanowires into hydrogels to create a tunable biointerface that could be readily integrated into any organ-on-a-chip and cell chip system. In the presented work, we investigated the matrix elasticity (Young's modulus, stiffness, adhesive force, and roughness) and nanotopography of gold nanowire loaded onto fibrin hydrogels using the bio-AFM (atomic force microscopy) method. Additionally, we investigated the capacity of human amniotic mesenchymal stem cells (hAMSCs) to differentiate into osteo- and chondrogenic lineages. Our results demonstrated that nanogold structured-hydrogels promoted differentiation of hAMSCs as shown by a significant increase in Collagen I and II production. Additionally, there was enhanced calcium mineralization activity and proteoglycans formation after a cultivation period of two weeks within microfluidic devices.

Targeted DNA delivery to cancer cells using a biotinylated chitosan carrier.

A novel biotinylated chitosan-graft-polyethyleneimine (Bio-Chi-g-PEI) copolymer was synthesized and evaluated as a nonviral gene delivery carrier for improvement of the transfection efficiency, endosomal escape, and targeted gene delivery of a plasmid encoding green fluorescent protein N1 (pEGFP-N1) into two different biotin-overexpressing cell lines including HeLa and OVCAR-3 cells. The structure of the obtained copolymers was confirmed by 1 H nuclear magnetic resonance (1 H NMR) and Fourier transform infrared spectroscopy. Physicochemical properties of the Bio-Chi-g-PEI/plasmid DNA (pDNA) complexes such as complex stability, size, zeta potential, and their morphology were investigated at various weight ratios of copolymer to pDNA. Bio-Chi-g-PEI copolymers could effectively condense pDNA into small particles with average diameters less than 164 nm and the zeta potential of +34.8 mV at the N/P ratio of 40/1. As revealed by flow cytometry, Bio-Chi-g-PEI/pDNA complexes had lower cytotoxicity than that of PEI 25 kDa/pDNA complexes in both cell lines. In vitro experiments revealed that the Bio-Chi-gPEI/pDNA complexes not only had much lower cytotoxicity, but also displayed higher transfection efficiency than that of PEI 25kDa/pDNA complexes. High percentage of cancer cells was successfully transfected by Bio-Chi-g-PEI/pDNA and properly expressed GFP protein. This study indicates that this copolymer complex can be a promising gene delivery carrier.