Spectrophotometric analysis of thrombolytic activity: SATA assay

Introduction: Measurement of thrombolytic activity i.e. clot lysis is crucial for research and development of novel thrombolytics. It is also a key factor in assessment of the effectiveness of conventionally used thrombolytic agents in the clinic, which are the choice effective therapies for myocardial infarction and ischemic stroke. Previous methods used for the assessment of thrombolytic activity are often associated with some drawbacks such as being costly, time-consuming, complication and low accuracy. Here, we introduce a simple, economic, relatively accurate and fast method of spectrophotometric analysis of thrombolytic activity (SATA) assay, standardized by tissue plasminogen activator (tPA), which can quantitatively measure in vitro thrombolytic activity. Methods: Blood clots were formed, uniformly, by mixing citrated whole blood with partial thromboplastin time (PTT) reagent, together with calcium chloride. Then, designated concentrations of tPA were added to the samples, and the released red blood cells from each clot were quantified using spectrophotometry (λmax= 405 nm) as an indicator of thrombolytic activity. The accuracy of the method was tested by assessment of dose-responsibility against R2 value obtained by linear equation and measurement of limit of detection (LOD) and limit of quantification (LOQ). The SATA assay was validated in comparison with some currently used techniques. Results: A linear relationship was obtained between different concentrations of tPA versus the spectrophotometric absorbance of the related dilutions of lysed clots, at λmax = 405 nm. Calculated R2 values were greater than 0.9; with LOD of 0.90 μg/mL of tPA (436.50IU) and LOQ of 2.99 μg/mL of tPA (1450.15IU). Conclusions: Conclusively, the SATA assay is a very simple quantitative method with repeatable and reproducible results for estimating the potency of an unknown thrombolytic agent, and calculating the activity as delicate as 1 μg/mL of tPA (485 IU/mL of thrombolytic dose). © 2018 The Author(s)

Aptamedicine: a new treatment modality in personalized cancer therapy

Aptamers (Aps) are short single-strand nucleic acids exhibiting unique 3D structure which facilitate their targeting potential against various cancer molecular markers (CMMs). Such features of Aps not only make them as suitable homing agents in targeted drug delivery systems (DDSs) but also candidate them as macromolecules that inhibit the interaction of the target ligand with other proteins. On the other hand, the conjugation of Aps with another therapeutic molecule such as antisense oligonucleotides (ASOs), siRNAs/miRNAs, Aps, toxins, chemotherapeutic agents, DNAzymes/Ribozymes provides hopeful strategy to eradicate the malignancies and overcome the off-target unwanted side effects. Such prominent features of Aps make them a promising treatment modality to overcome the tumor complexity and heterogeneity, which can be consequently applied for personalized therapy of cancer by using bispecific Ap-based therapeutics

Mesenchymal stem cell secretome induced the acquisition of anti-inflammatory phenotype in rat cortical microglia in vitro

Introduction: Here, we aimed to address the impact of mesenchymal stem cells-conditioned medium (MSCs-CM) on M1/M2 phenotype shifting of rat microglia after 48 hours in vitro. Methods: Rat neonatal cortical microglia were randomly allocated into four different groups as follows: Control; MSC-CM; IL-4; and Anti-IL-4 groups. In the MSC-CM, microglia were treated with MSC condition media. In the interleukin-4 (IL-4), cells received 20 ng/mL IL-4; conceived as a positive M2 control group). In the Anti-IL-4, the combination of IL-4 peptide and anti-IL-4 antibody was used. After 48 hours, protein levels of Iba1, CD86, and CD206 were monitored using immunofluorescence imaging and flow cytometry analysis. Results: According to immunofluorescence imaging, 48-hour incubation of rat cortical microglia with stem cells condition media increased protein levels of CD206 and decreased CD86, showing the polarization of microglia toward M2 type lineage compared to the non-treated control group. We found a similar trend in the group that received IL-4. By contrast, the incubation of microglia with anti-IL-4 antibody blunted the stimulatory effect of IL-4 to promote M2-type microglia. Similar to the immunofluorescence data, flow cytometry analysis revealed a significant increase of CD206 positive microglia after exposure to MSC-CM and IL-4 (P<0.05). Treatment with anti-IL-4 antibody significantly reduced the percent of CD206 positive cells, showing the inhibition of M1-to-M2 phenotype acquisition. Conclusion: The current study highlighted a notable anti-inflammatory effect of MSC secretome on cortical microglia by promoting M1-to-M2 phenotype acquisition

Combating atherosclerosis with targeted nanomedicines: recent advances and future prospective

Introduction: Cardiovascular diseases (CVDs) is recognized as the leading cause of mortality worldwide. The increasing prevalence of such disease demands novel therapeutic and diagnostic approaches to overcome associated clinical/social issues. Recent advances in nanotechnology and biological sciences have provided intriguing insights to employ targeted Nanomachines to the desired location as imaging, diagnosis, and therapeutic modalities. Nanomedicines as novel tools for enhanced drug delivery, imaging, and diagnosis strategies have shown great promise to combat cardiovascular diseases. Methods: In the current study, we intend to review the most recent studies on the nano-based strategies for improved management of CVDs. Results: A cascade of events results in the formation of atheromatous plaque and arterial stenosis. Furthermore, recent studies have shown that nanomedicines have displayed unique functionalities and provided de novo applications in the diagnosis and treatment of atherosclerosis. Conclusion: Despite some limitations, nanomedicines hold considerable potential in the prevention, diagnosis, and treatment of various ailments including atherosclerosis. Fewer side effects, amenable physicochemical properties and multi-potential application of such nano-systems are recognized through various investigations. Therefore, it is strongly believed that with targeted drug delivery to atherosclerotic lesions and plaque, management of onset and progression of disease would be more efficient than classical treatment modalities

Atherosclerosis preventive effects of marrubiin against (TNF-α)-induced oxidative stress and apoptosis

Introduction: Atherosclerosis is a complicated cascade of inflammatory processes, oxidative stress, and apoptosis, making it the most prevalent cardiovascular disease. The onset and progression of cardiovascular diseases are greatly influenced by oxidative stress. Targeting oxidative stress is an effective strategy for treating such diseases. Marrubiin is a bioactive furan labdane diterpenoid acts as a strong antioxidant to protect against oxidative damage. This study aimed to investigate the protective effects of marrubiin against oxidative stress and apoptosis in a cellular model of the vascular system. Methods: Human umbilical vein endothelial cells were treated with varying concentration of marrubiin and its IC50 value was determined. The antioxidant potential of marrubiin was assessed by measuring the intracellular level of glutathione (GSH) using a colorimetric technique. Since apoptosis plays a significant role in the plaque rupture, the study also evaluated the protective effects of marrubiin on the expression of key genes involved in apoptotic pathways. Results: Cells treated with marrubiin showed increased GSH levels compared to cell therapy control cells, indicating marrubiin’s ability to counteract the effects of TNF-α’s on GSH levels. Furthermore real-time PCR analysis demonstrated that marrubiin upregulated Bcl-xl while downregulating caspase3 and Nox4 in treated cells. These findings suggest that marrubiin protects against apoptosis and oxidative stress. Conclusion: Based on our findings, marrubiin is recommended as a preventive/therapeutic treatment for diseases caused by elevated intracellular reactive oxygen species levels in cardiovascular diseases

Electrosprayed Nanoparticles Containing Hydroalcoholic Extract of Echinacea purpurea (L.) Moench Stimulates Immune System by Increasing Inflammatory Factors in Male Wistar Rats

Purpose: Echinacea purpurea (L.) Moench is a member of the Asteraceae family and is traditionally used mainly due to its immunostimulatory properties. Various compounds including alkylamides and chicoric acid were reported as active ingredients of E. purpurea. Here, we aimed to prepare electrosprayed nanoparticles (NPs) containing hydroalcoholic extract of E. purpurea using Eudragit RS100 (EP-Eudragit RS100 NPs) to improve the immunomodulatory effects of the extract. Methods: The EP-Eudragit RS100 NPs with the different extract:polymer ratios and solution concentrations were prepared using the electrospray technique. The size and morphology of the NPs were evaluated using dynamic light scattering (DLS) and field emission-scanning electron microscopy (FE-SEM). To evaluate the immune responses, male Wistar rats were administrated with the prepared EP-Eudragit RS100 NPs and plain extract in the final dose of 30 or 100 mg/kg. The blood samples of the animals were collected and the inflammatory factors and complete blood count (CBC) were investigated. Results: In vivo studies indicated that the plain extract and EP-Eudragit RS100 NPs (100 mg/kg) significantly increased the serum level of tumor necrosis factor-α (TNF-α) and interleukin 1-β (IL1-β) whereas the EP-Eudragit RS100 NPs (30 mg/kg) significantly increased the number of white blood cells (WBCs) compared to the control group. Lymphocytes’ count in all groups was increased significantly compared to the control group (P<0.05) whereas other CBC parameters remained unchanged. Conclusion: The prepared EP-Eudragit RS100 NPs by electrospray technique caused significant reinforcement in the immunostimulatory effects of the extract of E. purpurea

Functional expression and impact of testis-specific gene antigen 10 in breast cancer: a combined in vitro and in silico analysis

Introduction: Testis-specific gene antigen 10 (TSGA10) is a less-known gene, which is involved in the vague biological paths of different cancers. Here, we investigated the TSGA10 expression using different concentrations of glucose under hypoxia and also its interaction with the hypoxia-inducible factor 1 (HIF-1). Methods: The breast cancer MDA-MB-231 and MCF-7 cells were cultured with different concentrations of glucose (5.5, 11.0 and 25.0 mM) under normoxia/hypoxia for 24, 48, and 72 hours and examined for the HIF-1α expression and cell migration by Western blotting and scratch assays. The qPCR was employed to analyze the expression of TSGA10. Three-dimensional (3D) structure and the energy minimization of the interacting domain of TSGA10 were performed by MODELLER v9.17 and Swiss-PDB viewer v4.1.0/UCSF Chimera v1.11. The UCSF Chimera v1.13.1 and Hex 6.0 were used for the molecular docking simulation. The Cytoscape v3.7.1 and STRING v11.0 were used for protein-protein interaction (PPI) network analysis. The HIF-1a related hypoxia pathways were obtained from BioModels database and reconstructed in CellDesigner v4.4.2. Results: The increased expression of TSGA10 was found to be significantly associated with the reduced metastasis in the MDA-MB-231 cells, while an inverse relationship was seen between the TSGA10 mRNA level and cellular migration but not in the MCF-7 cells. The C-terminal domain of TSGA10 interacted with HIF-1α with high affinity, resulting in PPI network with 10 key nodes (HIF-1α, VEGFA, HSP90AA1, AKT1, ARNT, TP53, TSGA10, VHL, JUN, and EGFR). Conclusions: Collectively, TSGA10 functional expression alters under the hyper-/hypo-glycemia and hypoxia, which indicates its importance as a candidate bio-target for the cancer therapy

Comet Assay: A Method to Evaluate Genotoxicity of Nano-Drug Delivery System

Introduction: Drug delivery systems could induce cellular toxicity as side effect of nanomaterials. The mechanism of toxicity usually involves DNA damage. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method for detecting strand damages in the DNA of a cell with applications in genotoxicity testing and molecular epidemiology as well as fundamental research in DNA damage and repair. Methods: In the current study, we reviewed recent drug delivery researches related to SCGE. Results: We found that one preference for choosing the assay is that comet images may result from apoptosis-mediated nuclear fragmentation. This method has been widely used over the last decade in several different areas. Overall cells, such as cultured cells are embedded in agarose on a microscope slide, lysed with detergent, and treated with high salt. Nucleoids are supercoiled DNA form. When the slide is faced to alkaline electrophoresis any breakages present in the DNA cause the supercoiling to relax locally and loops of DNA extend toward the anode as a ‘‘comet tail’’. Conclusion: This article provides a relatively comprehensive review upon potentiality of the comet assay for assessment of DNA damage and accordingly it can be used as an informative platform in genotoxicity studies of drug delivery systems

Formulation and Physicochemical Characterization of Lycopene-Loaded Solid Lipid Nanoparticles

Purpose: Lycopene belongs to the carotenoids that shows good pharmacological properties including antioxidant, anti-inflammatory and anticancer. However, as a result of very low aqueous solubility, it has a limited systemic absorption, following oral administration. Methods: Here, we prepared a stable lycopene-loaded solid lipid nanoparticles using Precirol® ATO5, Compritol 888 ATO and myristic acid by hot homogenization method with some modification. The size and morphological characteristics of nanoparticles were evaluated using Scanning Electron Microscopy (SEM). Moreover, zeta potential and dispersity index (DI) were measured using zeta sizer. In addition, encapsulation efficiency (EE%), drug loading (DL) and cumulative drug release were quantified. Results: The results showed that the size and DI of particles was generally smaller in the case of SLNs prepared with precirol when compared to SLNs prepared with compritol. Scanning electron microscopy (SEM) and particle size analyses showed spherical SLNs (125 ± 3.89 nm), monodispersed distribution, and zeta potential of −10.06 ± 0.08 mV. High EE (98.4 ± 0.5 %) and DL (44.8 ± 0.46 mg/g) were achieved in the case of nanoparticles prepared by precirol. The stability study of the lycopene-SLNs in aqueous medium (4 °C) was showed that after 2 months there is no significant differences seen in size and DI compared with the fresh formulation. Conclusion: Conclusively, in this investigation we prepared a stable lycopene-SLNs with good physicochemical characteristic which candidate it for the future in vivo trials in nutraceutical industries

The Construction of Chimeric T-Cell Receptor with Spacer Base of Modeling Study of VHH and MUC1 Interaction

Adaptive cell immunotherapy with the use of chimeric receptors leads to the best and most specific response against tumors. Chimeric receptors consist of a signaling fragment, extracellular spacer, costimulating domain, and an antibody. Antibodies cause immunogenicity; therefore, VHH is a good replacement for ScFv in chimeric receptors. Since peptide sequences have an influence on chimeric receptors, the effect of peptide domains on each other's conformation were investigated. CD3Zeta, CD28, VHH and CD8α, and FcgIIα are used as signaling moieties, costimulating domain, antibody, and spacers, respectively. To investigate the influence of the ligation of spacers on the conformational structure of VHH, models of VHH were constructed. Molecular dynamics simulation was run to study the influence of the presence of spacers on the conformational changes in the binding sites of VHH. Root mean square deviation and root mean square fluctuation of critical segments in the binding site showed no noticeable differences with those in the native VHH. Results from molecular docking revealed that the presence of spacer FcgIIα causes an increasing effect on VHH with MUC1 interaction. Each of the constructs was transformed into the Jurkat E6.1. Expression analysis and evaluation of their functions were examined. The results showed good expression and function