A Cationic Stearamide-based Solid Lipid Nanoparticle for Delivering Yamanaka Factors: Evaluation of the Transfection Efficiency.

Induced pluripotent stem cells (IPSC) are preferred as an alternative source for regenerative medicine, disease modeling, and drug screening due to their unique properties. As seen from the previous studies in the literature, most of the vector systems to transfer reprogramming factors are viral-based and have some well-known limitations. This study aims to develop a non-viral vector system for the transfection of reprogramming factors. Cationic stearamide lipid nanoparticles (CSLN) were prepared via the solvent diffusion method. The obtained CSLNs were used for the delivery of plasmid DNA (pDNA) encoding Oct3/4, Sox2, Klf4, and GFP to fibroblast cell lines. The optimization studies, for zeta potential and particle size of the conjugate, was performed to achieve high cell viability. CSLN63 with 36.5±0.06 mV zeta potential and 173.6±13.91 nm size was used for the transfection of Fibroblast cells. The transfection efficiency was observed by following GFP expression and was found as 70 %±0.11. The expression of the Oct4, Sox2, Klf4 was determined by RT-qPCR; an increase was observed after the 12th cycle in Klf4 (Ct averages: 13,41), Sox2 (Ct averages; 12,4), Oct4 (Ct average; 13,77). The tendency of colonization was observed. The upregulation efficiency of Oct4 and SSEA-1 with CSLN and another non-viral vector designed for the transportation of Yamanaka factors developed in our lab previously were compared with flow cytometer analysis.

3-Hydroxyhexanoate-based polycationic nanoparticle system for delivering reprogramming factors.

Aim: In this study, we aimed to develop a polycationic non-viral carrier for the delivery of the reprogramming factors to the L929 fibroblast cell.Methods: We have prepared (3-hydroxybutyrate-co-3-hydroxyhexanoate) PHBHHx-based nanoparticles with the solvent diffusion method. Cytotoxicity of PXNs was determined via MTT assay. Transfection efficiency was evaluated via screening GFP expression by fluorescence microscopy. The expression of reprogramming factors (Oct4, Klf4, and Sox2) was determined by RT-qPCR.Results: PXNs with 32.9 ± 0.41 mV zeta potential and 177.6 ± 0.80 nm size were used for transfection of L929 Fbroblast cells. The percentage of cell viability of PXN were between 91.8%(±2.9) and 42.1%(±1.3). The transfection efficiency was found as 71.6%(±3,5). According to RT-qPCR data, the rate of transfection factors was significantly increased after the 11th cycle compared to non-transfected cells. Based on these results, it can be concluded that newly developed PXN is thought to be an effective tool for reprogramming cells.

Development of biodegradable antibacterial cellulose based hydrogel membranes for wound healing.

Cellulose-based hydrogels have wide applications in tissue engineering and controlled delivery systems. In this study, chloramphenicol (CAP) loaded 2,3 dialdehyde cellulose (DABC) hydrogel membranes were prepared, characterized and their antibacterial efficacy was evaluated. Bacterial cellulose (BC) secreted by Acetobacter xylinum was modified to become DABC by oxidation via the sodium metaperiodate method. CAP-BC and CAP-DABC interactions were illustrated via ATR-FTIR analysis. Water retention capacity of BC and DABC membranes were determined as 65.6±1.6% and 5.3±0.3%, respectively. CAP release profiles were determined via HPLC analysis. The drug-loading capacities of BC and DABC membranes were 5mg/cm(2) and 0.1mg/cm(2), respectively. Membranes released 99-99.5% of the contained CAP within 24h and an initial burst release effect was not observed. In vitro antibacterial tests of BC and DABC, both CAP-loaded, demonstrated their ability to inhibit bacterial growth for a prolonged duration. Antimicrobial effect against bacteria was still prevalent after 3 days of incubation period with disc diffusion tests. The MTT test results reveal that fibroblast adhesion and proliferation on CAP-loaded DABC membranes were noticeably higher than CAP-loaded BC membrane. This newly developed drug containing DABC membranes seem to be highly suitable for wound healing due to its unique properties of biodegradability, biocompatibility, and antimicrobial effectiveness.

A thermo-sensitive NIPA-based co-polymer and monosize polycationic nanoparticle for non-viral gene transfer to smooth muscle cells.

Primary smooth muscle cells (SMC) isolated from the aorta of fetal calf were transfected with a green fluorescent protein (GFP)-encoding plasmid DNA, which was carried by a water-soluble and temperature-sensitive N-isopropylacrylamide-based (NIPAAm-based)-co-polymer, either poly(N-isopropylacrylamide-co-2-methacryloamidohistidine) (poly(NIPAAm-co-MAH)) or monosized PEGylated nanoparticle poly(styrene/poly(ethylene glycol) ethyl ether methacrylate/N-(3-(dimethylamino)propyl) methacrylamide) (poly(St/PEG-EEM/DMAPM)). Poly(NIPAAm-co-MAH) co-polymer was synthesized by solution polymerization of n-isopropylacrylamide (NIPAAm) and 2-methacrylamidohistidine (MAH). Monosized cationic nanoparticles were produced by emulsifier-free emulsion polymerization of styrene, PEG ethyl ether methacrylate and N-[3-(dimethyl-amino) propyl] methacrylamide, in the presence of a cationic initiator, 2,2-azobis (2-methylpropionamidine) dihydrochloride. The structure of poly(St/PEG-EEM/DMAPM) and poly(NIPAAm-co-MAH) was confirmed by(1) H-NMR and FT-IR spectroscopy. Particle size/size distribution and surface charges of both carriers were measured by Zeta Sizer. The LCST behavior of poly(NIPAAm-co-MAH) co-polymer was followed spectrophotometrically. Poly(St/PEG-EEM/DMAPM) nanoparticles, with an average size of 78 nm and zeta potential of 54.4 mV, and an average size of 200 nm with a zeta potential of 54.2 mV, and poly(NIPAAm-co-MAH) were used in the transfection studies. The cytotoxicity of the vectors was tested using the MTT method. According to conditions for the transfection study (polymer/cell ratio and polymer-cell incubation period), cell loss was only 4 and 15% with poly(St/PEG-EEM/DMAPM) sized 78 and 200 nm, respectively. Poly(NIPAAm-co-MAH) cytotoxicity was insignificant. Poly(NIPAAm-co-MAH) uptake efficiency in SMCs was around 85%, but gene expression efficiency were low compared to poly(St/PEG-EEM/DMAPM)/pEGFP-N2 conjugates because of the low zeta potential of the co-polymer. Polymer uptake efficiencies of the nanoparticles were 90-95%. GFP expression efficiency was 68 and 64% after transfection with pEGFP-N2 conjugate with 78 and 200 nm sized poly(St/PEG-EEM/DMAPM) nanoparticles.

Monosize polycationic nanoparticles as non-viral vectors for gene transfer to HeLa cells.

Monosized cationic nanoparticles were produced by emulsifier-free emulsion polymerization of styrene, poly(ethylene glycol) methacrylate and N-[3-(dimethyl-amino) propyl] methacrylamide, conducted in the presence of a cationic initiator, 2,2'-azobis (2-methylpropionamidine) dihydrochloride, using different amounts of ingredients and at different conditions. The structure of the terpolymers was confirmed by (1)H-NMR and FTIR spectroscopy. The nanoparticles, with an average size of 71.3 nm [polydispersity index (PDI), 1.110] and a Zeta potential of 65.6 mV obtained by a zeta sizer, were used in the transfection studies. HeLa cells were transfected in in vitro cell cultures with these non-viral nanoparticle vectors with a green fluorescent protein (GFP)-expressing plasmid DNA. The transfer of the cationic nanoparticles into the cells and GFP expressions with the conjugates of the nanoparticles and the GFP-expressing plasmid were followed by both light and fluorescent microscopy. The GFP expression efficiency was unexpectedly high (up to 90%).