Development of an anti-CD45RA-quantum dots conjugated scFv to detect leukemic cancer stem cells.

Leukemic cancer stem cells (LSCs), aberrantly overexpressing CD45RA are among the major causes of relapse following chemotherapy in patients with acute myeloid leukemia and serve as a highly sensitive marker for predicting relapse occurrence following chemotherapy. The main purpose of current study was to develop a sensitive approach for detecting LSCs based on a conjugate of an anti-CD45 scFv and quantum dot. The variable light and heavy chain sequences of a recently developed anti-CD45RA monoclonal antibody were derived from hybridoma cells and PCR amplified to construct scFv. Following insertion of scFv gene into a pET32a-lic vector and expression in Escherichia coli and purification, the purified scFv, was conjugated with carbon dots (C dots) and used for the detection of CD45RA +cells while CD45RA-cells served as negative control. Subsequently, Functional activity of the conjugate was analyzed by flow cytometry and ICC to detect the cell surface antigen binding and detection ability. Based on results, purified CD45RA scFv conjugated C dots could specifically recognize CD45RA positive cells, but not any CD45RA negative ones. In conclusion, here we developed a low-cost but very efficient approach for detection of CD45RA positive cells including LSCs.

Melatonin affects membrane integrity, intracellular reactive oxygen species, caspase3 activity and AKT phosphorylation in frozen thawed human sperm.

Cryopreservation is known to induce oxidative stress in spermatozoa. Although melatonin has powerful antioxidant properties, little is known about its effects on human sperm quality during cryopreservation. The present study was undertaken to investigate the effects of melatonin treatment on human sperm parameters essential for fertilization. We first evaluated the effects of various concentrations of melatonin (0-15 mM) on human sperm parameters such as motility, viability and levels of intracellular reactive oxygen species during cryopreservation in order to identify an optimal dose with the greatest effects for further studies. Liquefied semen samples were then divided into three aliquots: cryopreserved without melatonin (control), cryopreserved with 3 mM melatonin and fresh groups. After being thawed, samples were evaluated for motility, viability, membrane integrity, intracellular reactive oxygen species levels, caspase-3 activity and AKT phosphorylation. Treatment of spermatozoa with the various concentrations of melatonin significantly increased their motility and viability and decreased their intracellular reactive oxygen species levels compared with the control group. The optimal melatonin concentration (3 mM) significantly decreased the intracellular reactive oxygen species levels, caspase-3 activity and the percentage of both dead and apoptotic-like sperm cells and increased the vitality, progressive motility and total motility and AKT phosphorylation compared with the control group. Thus, melatonin exerts protective effects against cryodamage during human spermatozoa cryopreservation and may exert its effects via the PI3K/AKT signaling pathway.

Albumin coated copper-cysteine nanozyme for reducing oxidative stress induced during sperm cryopreservation.

In the present work, SOD mimetic nanozyme (NACu-Cys) consisting of Cu-Cys complex and nano-albumin (NA) were synthesized. After characterizing the nanozyme, its superoxide dismutase (SOD) behavior was evaluated by inhibition of the pyrogallol autoxidation method. The results revealed that NACu-Cys exhibited SOD mimetic activity with a half inhibition concentration (IC50) value of 7.0 × 10-3 µM and a turnover number (kcat) of 5.4 × 107 s-1. In the next step, this nanozyme was applied as a protective agent against oxidative stress induced by sperm cryopreservation. Increasing the motility, raising the viability and reducing the apoptosis occurred as a result of NACu-Cys additions to human sperm freezing medium. Comparison between the natural SOD and SOD mimic behavior of NACu-Cys revealed that this nanoparticle has the ability to be used as oxidative stress decrescent during cryopreservation process.

Cellular Therapy: The Hope for Covid-19.

Coronaviruses (CoVs) are a group of very diverse viruses that cause a broad spectrum of diseases from mild to severe enteric, respiratory, systemic diseases, and common cold or pneumonia among humans and animals. This virus is associated with Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and lung disease that lead to Acute Respiratory Distress Syndrome (ARDS). In December 2019, researchers identified a novel coronavirus type, called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), which was associated with symptoms of high fever, dry cough, headache, diarrhea, and reduction of White Blood Cells (WBC). Coronavirus-associated acute respiratory disease was named Coronavirus Disease 19 (COVID-19). No proven treatment has been discovered for COVID-19 so far, but researchers are trying to find the best effective way to treat this disease. Therefore, therapeutic strategies that facilitate the recovery of COVID-19 patients and reduce life-threatening complications are urgently needed now. Today, Mesenchymal Stem Cells (MSCs) and their secretion are utilized as one of the most applied tools to treat various diseases such as inflammation and cancer. MSC-derived vesicles are rich in various growth factors, cytokines, and interleukins that are produced and secreted under different physiological or pathological conditions. These vesicles were considered a suitable and effective tool in regeneration medicine because of their high power in repairing damaged tissues and modulating immune responses. Recently, evidence has shown MSC-derived vesicles through reduced expression of pro-inflammatory cytokines could improve damaged tissues in COVID-19 patients. In addition to MSCs and MSC-derived exosomes, Natural Killer (NK) cells, T cells, and platelet lysates were used against viral infection. In this review, we tried to provide an overview of MSC secretion and immune cells for COVID-19 therapy.

An electrochemical genosensor for differentiation of fully methylated from fully unmethylated states of BMP3 gene.

The methylation state of a part of the BMP3 gene was detected by our genosensor. This epigenetic biomarker is involved in the biomarker panel of the sDNA test, which is an FDA approved test for colorectal cancer screening. In the present genosensor, polyethyleneimine-stabilized silver nanoparticles (PEI-AgNPs) were used as a non-specific nanolabel for signal generation/amplification and lowering the limit of detection. After immobilization of capture probes and mercaptoethanol molecules on the gold electrode, a thermally treated mixture of the BMP3 targets and reporter probes was introduced to the electrode. Because of the specificity of the reporter probes for fully methylated targets, complete sandwich-like complexes are formed only with them. Therefore, such full-length double-stranded hybrids compared to fully unmethylated targets have more negative charges and can more attract positively charged PEI-AgNPs. For discrimination between methylated and unmethylated targets, electroimpedance spectroscopy and cyclic voltammetry were used for electrode modification monitoring and signal measurement. The sharp and narrow anodic peaks of cyclic voltammograms, which resulted from silver oxidation, were utilized for calibration plot analysis. The genosensor showed a linear response for the target concentration range from 1fM to 100 nM, while the detection limit for methylated and unmethylated target discrimination was 1 fM.

Zeolite-based nanocomposite as a smart pH-sensitive nanovehicle for release of xylanase as poultry feed supplement.

Xylanase improves poultry nutrition by degrading xylan in the cell walls of feed grains and release the entrapped nutrients. However, the application of xylanase as a feed supplement is restricted to its low stability in the environment and gastrointestinal (GI) tract of poultry. To overcome these obstacles, Zeozyme NPs as a smart pH-responsive nanosystem was designed based on xylanase immobilization on zeolitic nanoporous as the major cornerstone that was modified with L-lysine. The immobilized xylanase was followed by encapsulating with a cross-linked CMC-based polymer. Zeozyme NPs was structurally characterized using TEM, SEM, AFM, DLS, TGA and nitrogen adsorption/desorption isotherms at liquid nitrogen temperature. The stability of Zeozyme NPs was evaluated at different temperatures, pH, and in the presence of proteases. Additionally, the release pattern of xylanase was investigated at a digestion model mimicking the GI tract. Xylanase was released selectively at the duodenum and ileum (pH 6-7.1) and remarkably preserved at pH ≤ 6 including proventriculus, gizzard, and crop (pH 1.6-5). The results confirmed that the zeolite equipped with the CMC matrix could enhance the xylanase thermal and pH stability and preserve its activity in the presence of proteases. Moreover, Zeozyme NPs exhibited a smart pH-dependent release of xylanase in an in vitro simulated GI tract.

Gold Nanoparticles Conjugated L-Lysine for Improving Cisplatin Delivery to Human Breast Cancer Cells.

INTRODUCTION: Nano drug delivery is a broad field of research on the development of novel nano- carrier systems for effective therapeutic delivery of drugs. Here, an anticancer drug, cisplatin (CDDP) conjugated Gold Nanoparticles (GNPs) via L-Lysine (Lys) linker. METHODS: The produced nanodrug (GNPs-Lys-CDDP) was characterized by UV-Vis spectroscopy, Dynamic Light Scattering (DLS), Zeta potentials and electron force microscopy. The cytotoxic efficacy of the GNPs-Lys-CDDP against human breast cancer cells (SKBR3) and normal cells (MCF- 10A) was evaluatedby MTT assay. Cell apoptosis and morphology changes were assessed by flowcytometery and Acridine Orange/Ethidium Bromide (AO/EtBr) staining, respectively. RESULTS: It was found that the GNPs-Lys-CDDP with a size of 85 nm and negatively charged with a zeta-potential of about -25 mV could be taken up by tumor cells. A marked change in the UV spectrum of GNPs-Lys-CDDP compare to GNPs showed a strong absorption shift in the 525 nm region. The LD 50 of GNPs-Lys-CDDP against SKBR3 (1 µg.mL -1), was found to be 8 times lower than that of naked CDDP against SKBR3 (8 µg.mL -1). The nanocomplex GNPs-Lys-CDDP also significantly increased the apoptosis of SKBR3 with the lowest cytotoxic effects on normal cells. DISCUSSION: This work indicates that GNPs effectively could decrease the lethal dose of CDDP to 87%. Hence, GNPs modified by Lys, could be a good nano-carrier for chemotherapeutic drugs.

Silver-gold-apoferritin nanozyme for suppressing oxidative stress during cryopreservation.

Reactive oxygen species (ROS) cause oxidative stress, which involves in the pathogenesis of many serious diseases. Apoferittin containing gold-silver nanoparticles (Au-Ag-AFT) was designed and evaluated as a nanozyme for scavenging the ROS. The nanozyme consisting of silver-gold nanohybrid in apoferittin cage represents superoxide dismutase, catalase and peroxidase mimetic activities. The Au-Ag-AFT nanozyme was characterized by spectroscopy, FESEM, TEM and dynamic light scattering. The inhibition process for pyrogallol autoxidation was used for assaying the superoxide dismutase mimetic activity and measuring the kinetic parameters of Au-Ag-AFT nanozyme. Additionally, Aebi method and standard protocol was used for evaluating the catalase and peroxidase mimetic activity. The kcat values for superoxide dismutase, catalase and peroxidase mimetics activity were 1.4 × 106, 0.1 and 9 × 103 s-1 respectively. These values indicated that Au-Ag-AFT nanozyme could act as a suitable ROS scavenger. Additionally, Au-Ag-AFT nanozyme was examined as a protective agent for human sperm against oxidative stress induced during the cryopreservation process. Presence of the nanozyme in the sperm media significantly increased the motility and viability of the cells and also decreased the ROS, apoptosis and necrosis (P < 0.05) compare to the control group.

Cancer diagnosis using nanomaterials based electrochemical nanobiosensors.

Cancer is one of the most important causes of mortality in the world, which can be severely reduced by early detection to avoid future problems in the field of economics and mental health. Hence, electrochemical nanobiosensors as portable devices for rapid detection of  cancer biomarkers, have found an important place in clinical medicine for diagnosis, managements or cancer screening. Although, these biosensors have been receiving attention in the recent years, their principles are unchanged. By progress in nanotechnology, a great potential has been giving to nanobiosensors. Applications of a wide variety of nanomaterials in developing electrochemical biosensors, led to the production of potential nanobiosensors. Due to the high electrical conductivity, and increased surface area relative to the volume along with more repeatability, the application of NPs in electrochemical biosensors has been developed. Therefore, in this review, we discussed the impact of nanomaterials on the accuracy of biosensors in early cancer detection such as lung, prostate, breast, and other cancers. However, the modification of electrode performance by nanomaterials is relatively complicated, which causes limitation for some nanomaterials to be used inbiosensor applications. Indeed, the construction of electrodes based on nanomaterial requires a simple, reliable and inexpensive route to increase the sensitivity and reproducibility. Thus, the aim of this study can be defined as determining the detection limit of electrochemical nanobiosensors as well as introducing the challenges of fabricating and designing electrochemical nanobiosensors based on nanomaterials and their evaluations in the future medical setting.

A genosensor for detection of HTLV-I based on photoluminescence quenching of fluorescent carbon dots in presence of iron magnetic nanoparticle-capped Au.

Carbon dots and Fe3O4@Au were synthesized to develop a new biosensor to detect DNA target. We investigated the photoluminescence property of carbon dots (CDs) in the presence of Fe3O4-capped Au (Fe3O4@Au). Firstly, we designed two dedicated probes for unique long sequence region of human T-lymphotropic virus type 1 genome. One of the probes was covalently bound to the CDs. In the absence of target, CDs-probe was adsorbed on the surface of Fe3O4@Au through two possible mechanisms, leading to quenching the fluorescence emission of CDs. The fluorescence emission of CDs was recovered in the presence of target since double-stranded DNA cannot adsorb on the Fe3O4@Au. Also, Fe3O4@Au can adsorb the unhybridized oligonucleotides and improves the accuracy of detection. The specificity of the proposed biosensor was confirmed by BLAST search and assessed by exposing the biosensor to other virus targets. The experimental detection limit of the biosensor was below 10 nM with linear range from 10 to 320 nM.

Cytotoxic effect of albumin coated copper nanoparticle on human breast cancer cells of MDA-MB 231.

PURPOSE: The aim of this study was to design a new nanocomposite that would have high cytotoxicity against invasive breast cancer cells and minimum side effects on normal cells. METHODS: An albumin nano-carrier for delivery of CuNPs was developed. The ACuNPs formation was characterized by TEM, DLS and UV-Vis, fluorescence and circular dichroism spectroscopy. The cytotoxic efficacy of the ACuNPs against human breast cancer cells (MDA-MB 231) and normal cells (MCF-10A) was compared using a standard MTT assay. The mechanism of cell death induced by ACuNPs was considered by inverted and fluorescent microscopy, flow cytometry and gel electrophoresis. The effects of compounds on ROS generations in MDA-MB 231 cells were also studied. RESULTS: It was found that the resulted ACuNPs with a diameter of 62.7 nm and zeta potential of about -10.76 mV, are suitable for extravasation into tumor cells. In ACuNPs, the 90% of the secondary structure and almost all the tertiary structure of albumin remained intact. Comparing to CuNPs, ACuNPs could significantly suppress the viability of cancer cells while they were less toxic on normal cells. Compared with the untreated cells, the MDA-MB 231 cell line showed higher levels of ROS production after treatment with ACuNPs. The increase in ROS production after 24 hours indicated that ACuNPs induce apoptosis. CONCLUSIONS: The ACuNPs characteristics such as intact structure of albumin, high toxicity against cancer cells comparing to normal cells and apoptosis induction as the mechanism of cell death, revealed that this nanocomposite is a good candidate to be used as a chemotherapeutic agent against invasive breast cancer cells.