Specific delivering of RNAi using Spike's aptamer-functionalized lipid nanoparticles for targeting SARS-CoV-2: A strong anti-Covid drug in a clinical case study.

Coronavirus (SARS-CoV-2) as a global pandemic has attracted the attention of many scientific centers to find the right treatment. We expressed and purified the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and specific RBD aptamers were designed using SELEX method. RNAi targeting nucleocapsid phosphoprotein was synthesized and human lung cells were inoculated with aptamer-functionalized lipid nanoparticles (LNPs) containing RNAi. The results demonstrated that RBD aptamer having KD values of 0.290 nm possessed good affinity. Based on molecular docking and efficacy prediction analysis, siRNA molecule was showed the best action. LNPs were appropriately functionalized by aptamer and contained RNAi molecules. Antiviral assay using q-PCR and ELISA demonstrated that LNP functionalized with 35 µm Apt and containing 30 nm RNAi/ml of cell culture had the best antiviral activity compared to other concentrations. Applied aptamer in the nanocarrier has two important functions. First, it can deliver the drug (RNAi) to the surface of epithelial cells. Second, by binding to the SARS-CoV-2 spike protein, it inhibits the virus entrance into cells. Our data reveal an interaction between the aptamer and the virus, and RNAi targeted the virus RNA. CT scan and the clinical laboratory tests in a clinical case study, a 36-year old man who presented with severe SARS-CoV-2, demonstrated that inhalation of 10 mg Apt-LNPs-RNAi nebulized/day for six days resulted in an improvement in consolidation and ground-glass opacity in lungs on the sixth day of treatment. Our findings suggest the treatment of SARS-CoV-2 infection through inhalation of Aptamer-LNPs-RNAi.

Ipomoea batatas: papain propeptide inhibits cysteine protease in main plant parasites and enhances resistance of transgenic tomato to parasites.

Different parasites cause severe lose in quantity and quality of crops. Many parasites develop haustorial cells and stylets that penetrate the host using secreted enzymes and mechanical pressure. Cysteine proteases are pre-pro-enzyme produced by parasites that are essential for normal parasitism. Papain is also a kind of cysteine proteases such that its propeptide segment has inhibitory properties and limits the protease activity of papain. To investigate the inhibitory effects of papain propeptide on some parasite proteases, we cloned inhibitory propeptide of papain of Ipomoea batatas, and enzymatic fragments of Diabrotica virgifera cathepsin L-like protease-1, Meloidogyne incognita cathepsin L-like protease 1, Heterodera glycines cysteine protease-1, Cuscuta chinesis cysteine protease and Orobanche cernua cysteine protease. After purification of recombinant inhibitory propeptide and enzymatic fragments, the inhibition activity of propeptide on cysteine proteases was measured. Finally inhibitory propeptide was transformed into tomato and transgenic plants resistance to parasites (bioassay) were examined. We demonstrated papain-propeptide inhibits cysteine protease of mentioned parasites. In transgenic tomato plants, papain-inhibitory propeptide effectively interrupted haustoria development. Haustoria-digitate cells of dodder could not differentiate and develop into the phloem and xylem hyphae on transgenic tomatoes. Parasites grown on transgenic tomatoes showed reduction in vigor and productivity due to defective connection of haustoria. Lower ratio of female nematodes and a decrease of nematode egg mass per transgenic line indicated biocontrol of nematode. The changes in growth factors of parasite challenged transgenic lines relative to controls, indicates the efficacy of papain propeptide in control of parasitism.

Fabrication of an immunosensor for early and ultrasensitive determination of human tissue plasminogen activator (tPA) in myocardial infraction and breast cancer patients.

Sensitive detection of biomarkers will mean accurate and early diagnosis of diseases. A tissue plasminogen activator (tPA) has a crucial role in many cardiovascular diseases and it is related to many processes such as angiogenesis in cancer cells. Therefore, sensitive determination of tPA is important in diagnosis and clinical research. tPA monoclonal antibody was covalently attached onto single-wall carbon nanotubes (SWCNTs) using diimide-activated imidation coupling. Functionalized SWCNTs were immobilized onto a glassy carbon electrode and the modification process was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SEM, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Cyclic voltammograms (CVs) in a scan rate of 100 mVs-1 was studied and comparisons were made between the modified glassy carbon electrodes (immobilized with antibodies) as a working electrode before and after the formation of tPA-antibody complex. Results of the SDS-PAGE demonstrated that the antibody was covalently and site directly attached to the SWCNTs. The fabricated biosensor provided a good linear response range from 0.1 to 1.0 ng mL-1 with a low detection limit of 0.026 ng mL-1. The immunosensor showed selectivity, reproducibility, good sensitivity, and acceptable stability. Satisfactory results were observed for early and sensitive determination of tPA in human serum samples. For the first time, such specific biosensor is currently being fabricated for tPA in our laboratories and successfully could determine tPA in myocardial infraction and breast cancer patients. Graphical abstract Fabricated biosensor for determination of tPA.

A label-free electrochemical biosensor based on tubulin immobilized on gold nanoparticle/glassy carbon electrode for the determination of vinblastine.

Vinblastine (VLB) is prescribed for a wide variety of cancers. Therefore, development of sensitive methods for early diagnosis is urgently required. In this work, a highly sensitive and label-free impedimetric biosensor was fabricated for the electrochemical detection of VLB. First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of a glassy carbon electrode (GCE). 3-Mercaptopropionic acid (MPA) was self-assembled over the AuNPs. Then, tubulin (TUB), as a receptor, was covalently immobilized at the AuNPs/GCE surface via carbodiimide coupling reaction using N-(3 dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) and N-hydroxy succinimide (NHS). The step-by-step modification process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of a redox probe [Fe(CN)6]3-/4-. The VLB concentration was measured through the increase of impedance values in the corresponding specific binding of VLB and TUB. The increased electron-transfer resistance (R et) values were proportional to the value of VLB concentrations in the range of 0.4 to 65.0 nmol L-1 with a detection limit of 8.4 × 10-2 nmol L-1 (SN-1 = 3). The practical analytical performance of the proposed method was demonstrated by determination of VLB in plant extracts and human serum samples with satisfactory recoveries.