Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells.

Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.

Sulfanilamide Modified Magnetic Nanoparticles for Purification of Carbonic Anhydrase from Bovine Blood.

Magnetic nanoparticles (MNPs) have been used for purification of specific biomolecules form mixtures. The aim of this study is to develop a new, cheap, reusable, and magnetic-based material to purify the carbonic anhydrase (CA) enzyme in a short time with high efficiency. In the first part of this study, silica-coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) were obtained. Surface modification of Fe3O4@SiO2 MNPs was accomplished with 3-(4-Hydroxyphenyl) propionic acid (PA) and sulfanilamide (SA), respectively. SA is a selective inhibitor of CA, and it selectively binds to CA. The final particle was named Fe3O4@SiO2-PA-SA MNPs and characterized by SEM, TEM, XRD, and FT-IR. It was determined that the produced MNPs contained multicore, were smaller than 100 nm in size, and had a spherical morphology. The CA was purified from bovine blood hemolysate in a short time such as 2.5 h and in a simple manner. The maximum enzyme purifying capacity of MNPs was calculated as 13.87 ± 3.27 mg CA/g MNP. SDS-PAGE analysis was confirmed that high CA purification success was achieved.

Comparative heating efficiency and cytotoxicity of magnetic silica nanoparticles for magnetic hyperthermia treatment on human breast cancer cells.

Magnetic hyperthermia (MHT) is a promising treatment for a variety of cancers due to its ability to increase the sensitivity of cells to other treatments, such as chemotherapy. Superparamagnetic nanoparticles (MNPs) were used for MHT treatment due to their heat generation ability under an AC magnetic field (AMF). In this study, iron oxide and zinc-doped iron oxide MNPs were produced and modified with silica to obtain eleven different types (MSNP-I to -XI) of magnetic silica nanoparticles (MSNPs). The MSNPs which show the highest heating capacity were selected to investigate their MHT ability on non-tumourigenic MCF-10A and tumourigenic MCF-7 cell lines. The cytotoxicity results indicated that the size, the content of the magnetic core and silica coating thickness were important in the heating capacity of MSNPs under AMF. After MHT treatment, selected MSNPs showed limited cytotoxicity on MCF-10A, but significant cell death on MCF-7. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03377-y.

Electrically controlled mRNA delivery using a polypyrrole-graphene oxide hybrid film to promote osteogenic differentiation of human mesenchymal stem cells.

Direct messenger ribonucleic acid (mRNA) delivery to target cells or tissues has revolutionized the field of biotechnology. However, the applicability of regenerative medicine is limited by the technical difficulties of various mRNA-loaded nanocarriers. Herein, we report a new conductive hybrid film that could guide osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) via electrically controlled mRNA delivery. To find optimal electrical conductivity and mRNA-loading capacity, the polypyrrole-graphene oxide (PPy-GO) hybrid film was electropolymerized on indium tin oxide substrates. We found that the fluorescein sodium salt, a molecule partially mimicking the physical and chemical properties of mRNAs, can be effectively absorbed and released by electrical stimulation (ES). The hADMSCs cultivated on the PPy-GO hybrid film loaded with pre-osteogenic mRNAs showed the highest osteogenic differentiation under electrical stimulation. This platform can load various types of RNAs thus highly promising as a new nucleic acid delivery tool for the development of stem cell-based therapeutics. Electronic Supplementary Material: Supplementary material (electrochemical and FT-IR analysis on the film, additional SEM, AFM and C-AFM images of the film, optical and fluorescence images of cells, and the primers used for RT-qPCR analysis) is available in the online version of this article at 10.1007/s12274-022-4613-y.

Synthesis, Characterization, Biological Activity and Molecular Docking Studies of Novel Organotin(IV) Carboxylates.

Four new carboxylates complexes with general formula R2SnL2 and R3SnL, where R = n-butyl (1, 3), methyl (2, 4) and L = 4-Chlorophenoxyacetate, were synthesized in significant yields. FT-IR analysis revealed a chelating (1 and 2) and a bridging bidentate (3 and 4) coordination modes for the carboxylate ligand in solid state which was further confirmed by the single crystal X-ray analysis of complex 4. The NMR data (1H, 13C and 119Sn) revealed a higher coordination number around the tin center in R2SnL2 (1 and 2) compared to R3SnL (3 and 4). A close matching was observed between the experimental and calculated structures (obtained at B3LYP/6-31G* + LANL2DZ basis set). Quantum chemical analysis indicates that the carboxylate moiety has the major contribution in the formation of filled and unfilled orbitals as well as in ligand to ligand intramolecular charge transfer during the electronic transitions. The cytotoxicity data of the screened compounds evaluated against lung cancer cell line (A549) and normal lung fibroblast cell line (MRC-5) revealed that 1, 3 and 4 have shown dose dependent cytotoxic effects while HL and 2 have shown steady and low cytotoxic activities. The antibacterial activity of complexes 1-4 is higher than that of HL. Molecular docking study showed an intercalation binding mode for complex 3 with DNA (docking score = -3.6005) involving four polar interactions. Complex 3 docking with tubulin (PDB ID 1SA0) with colchicine as a target protein resulted in three polar interactions (docking score -5.2957). Further, the docking analysis of the HL and 1-4 has shown an adequate interactions with the coronavirus SARS-CoV-2 spike protein, nucleocapsid protein and human angiotensin converting enzyme (ACE2).

Magnetic nanoparticle mediated-gene delivery for simpler and more effective transformation of Pichia pastoris.

The introduction of exogenous DNA into a cell can be used to produce large quantities of protein. Here, we describe a novel gene delivery method for Pichia pastoris based on recombinant DNA delivery using magnetic nanoparticles (MNPs) under magnetic forces. For this purpose, a linear plasmid (pGKB-GFP) containing the Green Fluorescent Protein (GFP) gene is loaded on polyethyleneimine-coated iron oxide (Fe3O4@PEI) MNPs at doses that are non-toxic to the yeast cells. The pGKB-GFP loaded MNPs combined with enhancer PEI (Fe3O4@PEI + pGKB-GFP + PEI) are directly transferred to non-competent cells. An effective GFP expression was observed by the selection of antibiotic-resistant yeast cells and heterologous gene integration into the P. pastoris genome was provided. This method, which is very simple, effective, and advanced equipment-free compared to traditional methods, uses smaller amounts of DNA and the process can be performed in a shorter time. The suggested method might also be adapted for the transformation of other yeast species.

Disulfiram-loaded functionalized magnetic nanoparticles combined with copper and sodium nitroprusside in breast cancer cells.

Disulfiram (DSF), one of the members of the dithiocarbamate family, is a reactive species (RS) generator and is capable of inducing cancer cell death in breast cancer. However, it is hydrophobic and highly degradable in blood. Therefore, drug delivery systems would be of great benefit in supporting the selective accumulation of DSF in tumor cells. In this study, it was aimed to prepare a drug carrier system based on magnetic mesoporous silica nanoparticles (Fe3O4@mSiO2 MNPs) which are non-toxic, biocompatible, and have a mesoporous structure. The Fe3O4@mSiO2 MNPs were modified with folic acid linked polyethyleneimine (PEI-FA) to increase both their solubility in water and specificity for cancer cells. Thus, the cancer-selective DSF-carrier system (mMDPF) was synthesized with a high surface area but with dimensions of less than 160 nm, and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and Brunauer-Emmett-Teller (BET) analysis. The drug-loading capacity of mMDPF was measured as 4.35% by high-performance liquid chromatography (HPLC) and the best drug release kinetics of mMDPF was observed at 37 °C and pH 6.0 which is the pH in the endosome. The cytotoxicity of the mMDPF on breast cancer (MCF-7) cells was improved by applying mMDPF with copper and/or sodium nitroprusside. It was observed that mMDPF was taken up more by MCF-7 cells and its toxicity on MCF-7 cells was much higher than non-tumorigenic (MCF-10A) cells.

Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins.

Conserved lipid transfer proteins of the Ups/PRELI family regulate lipid accumulation in mitochondria by shuttling phospholipids in a lipid-specific manner across the intermembrane space. Here, we combine structural analysis, unbiased genetic approaches in yeast and molecular dynamics simulations to unravel determinants of lipid specificity within the conserved Ups/PRELI family. We present structures of human PRELID1-TRIAP1 and PRELID3b-TRIAP1 complexes, which exert lipid transfer activity for phosphatidic acid and phosphatidylserine, respectively. Reverse yeast genetic screens identify critical amino acid exchanges that broaden and swap their lipid specificities. We find that amino acids involved in head group recognition and the hydrophobicity of flexible loops regulate lipid entry into the binding cavity. Molecular dynamics simulations reveal different membrane orientations of PRELID1 and PRELID3b during the stepwise release of lipids. Our experiments thus define the structural determinants of lipid specificity and the dynamics of lipid interactions by Ups/PRELI proteins.

Curcumin on the "flying carpets" to modulate different signal transduction cascades in cancers: Next-generation approach to bridge translational gaps.

Curcumin, a bioactive and pharmacologically efficient component isolated from Curcuma longa has attracted considerable attention because of its ability to modulate diverse cellular and physiological pathways. WNT, TGF/SMAD, NOTCH, and SHH are fundamentally different signaling cascades, but their choreographed activation is strongly associated with cancer development and progression. In this review we have attempted to set spotlight on regulation of different cell signaling pathways by curcumin in different cancers. We partition this multi-component review into in-depth biological understanding of various signal transduction cascades and how curcumin targets intracellular signal transducers of deregulated pathways to inhibit cancer development and progression. Rapidly broadening landscape of both established and candidate oncogenic driver mutations identified in different cancers is a major stumbling block in the standardization of drugs having significant clinical outcome. Intra and inter-tumor heterogeneity had leveraged the complexity of therapeutic challenges to another level. Multi-pronged approach and molecularly guided treatments will be helpful in improving the clinical outcome.