Fluorescence intensity was measured using a Synergy Mx microplate reader (BioTek Devices) at an excitation wavelength of 535?nm and emission wavelength of 595?nm

Fluorescence intensity was measured using a Synergy Mx microplate reader (BioTek Devices) at an excitation wavelength of 535?nm and emission wavelength of 595?nm. was stable for several days with no aggregation. X-ray photoelectron spectroscopy (XPS) validated their ternary composition, and it elucidated the 4-Methylumbelliferone (4-MU) molecular interactions among Ca2+, the plasmid DNA, and the AlgS. Efficient cellular NAV3 uptake ( 80%), associated with potent GFP gene expression (22%C35%), was observed across multiple cell types: main rat neonatal cardiac fibroblasts, human breast malignancy cell collection, and human hepatocellular carcinoma cells. The uptake mechanism of the NPs was analyzed using imaging circulation cytometry and shown to be via active, clathrin-mediated endocytosis, as chemical inhibition of this pathway significantly reduced EGFP expression. The NPs were cytocompatible and did not activate the 4-Methylumbelliferone (4-MU) T lymphocytes in human peripheral blood mononuclear cells. Proof of concept for the efficacy of these NPs as a carrier in malignancy gene therapy was exhibited for Diphtheria Toxin Fragment A (DT-A), resulting in abrogation of protein synthesis and cell death in the human breast malignancy cell collection. Collectively, our results show that this developed AlgS-Ca2+-plasmid DNA (pDNA) NPs may be used as an effective non-viral carrier for pDNA. influence of AlgS-Ca2+-pDNA NPs on peripheral blood mononuclear cells (PBMCs) from healthy individuals, exposing their effect on T?cell activation and cytokine production. Ultimately, the protein expression induced by the developed platform for model and therapeutic pDNA, across multiple cell types, was evaluated. Results Physico-chemical Characterization of the AlgS-Ca2+-pDNA NPs The assembly into NPs by electrostatic interactions among Ca2+, pDNA, and AlgS was validated in high-resolution transmission electron microscopy (TEM) images (the final concentrations of components were 2.5?g/mL AlgS, 25?mM Ca2+, and 15?ng/L pDNA for dry-TEM and 25?g/mL AlgS, 250?mM Ca2+, and?150?ng/L pDNA for cryogenic-TEM [cryo-TEM]) (Physique?1). The NP size, measured on images from cryo-TEM, showed particles?with a mean diameter of 188? 50 (n?= 17), much larger than the size observed in the dry-TEM images, indicating that water molecules participate in the assembly and structure of these?NPs. Open in a separate window Physique?1 High-Resolution TEM Images of AlgS-Ca2+-pDNA NPs (A and B) Dry-TEM micrographs of NPs (2.5?g/mL AlgS, 25?mM Ca2+, and 15?ng/L pDNA) with gold-labeled AlgS. (C) Cryo-TEM micrographs of complexes (250?mM Ca2+ and 150?ng/L pDNA). (D) Cryo-TEM micrographs of NPs (25?g/mL AlgS, 250?mM Ca2+, and 150?ng/L pDNA). Level bars, 500?nm (A) and 100?nm (BCD). The dynamic light 4-Methylumbelliferone (4-MU) scattering (DLS) analysis of the NPs (diluted 1:50) reveals a mean hydrodynamic diameter of 270?nm (Table 1), slightly larger than the size directly measured around the TEM images. This difference?could be due to the different methods utilized for the analysis;?in DLS, the assumption is that particles are spherical, while the TEM?images show the NPs are not perfectly that. Most notably, the size of?the AlgS-Ca2+-pDNA NPs was nearly twice the size of AlgS-Ca2+-siRNA NPs (130?nm15), as expected due to the larger size of pDNA. Table 1 Size Distribution and Surface Charge of NPs Prepared with Different Concentrations of Ca2+ over 72 h and for future gene therapy. Materials and Methods Materials and Cells The plasmids pEGFP N1 (4,733?bp, GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”U55762″,”term_id”:”1377911″,”term_text”:”U55762″U55762) and pGL3 (4,818?bp, GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”U47298″,”term_id”:”13195706″,”term_text”:”U47298″U47298) were kindly provided by Professor Ziv?Reich (Weizmann Institute of Science, Israel). Labeling of plasmids with fluorescein or Cy5, using Label IT Tracker (fluorescein or Cy5)?Nucleic Acid Labeling Kit (Mirus Bio, Madison WI), was performed according to the manufacturers instructions. The DT-A- (UniProtKB: “type”:”entrez-protein”,”attrs”:”text”:”Q6NK15″,”term_id”:”81402020″,”term_text”:”Q6NK15″Q6NK15) encoding plasmid, pDT-A N1 (4,671?bp), was designed by replacing the GFP gene from pEGFP N1 with DT-A. Based on the sequence provided by us, the DT-A gene was synthesized by Syntezza Bioscience (Jerusalem, Israel) and sub-cloned by Bio Basic (Markham, ON, Canada). All plasmids were propagated in and purified?by QIAGEN Midiprep packages according to the manufacturers instructions (Hilden, Germany). Dynabeads Human T-Activator CD3 and 4-Methylumbelliferone (4-MU) CD28 were used according to 4-Methylumbelliferone (4-MU) the manufacturers instructions (Thermo Fisher Scientific, MA, USA). All antibodies used.