Chondrogenesis of human mesenchymal stem cells by microRNA loaded triple polysaccharide nanoparticle system

WOS: 000472241700072PubMed ID: 31147048Degenerative cartilage is the pathology of severe depletion of extracellular matrix components in articular cartilage. In diseases like osteoarthritis, misregulation of microRNAs contributes the pathology and collectively leads to disruption of the homeostasis. In this study chondroitin sulfate/hyaluronic acid/chitosan nanoparticles were prepared and successfully characterized chemically and morphologically. Results demonstrated higher chondroitin sulfate amounts led smaller nanoparticles, but lower surface zeta potential due to high electronegativity. After optimization of chondroitin sulfate amounts regarding size and charge, nanoparticles were loaded with microRNA-149-5p, a therapeutic miRNA downregulated in osteoarthritis, and evaluated focusing on their loading efficiency, release behaviour, cytotoxicity and gene transfection efficiency in vitro. Results showed all nanoparticle formulations were non-toxic and promising gene delivery agents, due to increased levels of microRNA-149-5p and decreased mRNA levels of microRNA's target, FUT-1. Highest gene transfection efficiency was obtained with the nanoparticle formulation which had the highest chondroitin sulfate load and smallest size. In addition, owing to their high chondroitin sulfate cargo, all nanoparticles were reported to enhance chondrogenesis, which was demonstrated by gene expression analysis and sulfated glycosaminoglycan (sGAG) staining. The obtained data suggest that the delivery of microRNA-149-5p via polysaccharide based carriers could achieve collaborative impact in cartilage regeneration and have a potential to enhance osteoarthritis treatment

EFFECTS OF ABCE1 DOWN-REGULATION BY RNAi ON CHEMOSENSITIVITY OF LUNG CANCER CELLS

WOS: 000343139500383

Downregulation of ABCE1 via siRNA affects the sensitivity of A549 cells against chemotherapeutic agents

WOS: 000351474100017PubMed: 25744244ATP-binding cassette E1 (ABCE1) is involved in several biological functions in cancer cells such as tumor proliferation, antiapoptotic pathway and chemoresistance mechanism. This work aimed to investigate the alterations in chemosensitivity of A549 lung cancer cells for 5-Fluorouracil (5-FU) and irinotecan by silencing ABCE1 using specific small interfering RNAs (siRNA). The cells were treated with low doses of drugs, alone and also their combinations with ABCE1 siRNA. Cytotoxicity, cell proliferation and apoptosis/necrosis evaluations were performed in order to examine the effects of the combined treatment. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to confirm the downregulation of ABCE1. We also investigated the levels of B cell lymphoma 2 (Bcl-2) and mammalian target of rapamycin (mTOR) after the treatments by RT-PCR. Downregulation of ABCE1 improved the anticancer effects of 5-FU in inducing cell viability/proliferation inhibition and apoptosis/necrosis, whereas interestingly, almost did not change or slightly reduced the anticancer effects of irinotecan. ABCE1 expression significantly decreased by transfecting the cells with ABCE1 siRNA. Moreover, Bcl-2 and mTOR levels changed after the single or combined therapy in parallel with the apoptotic and antiproliferation effect. In conclusion, the simultaneous treatment of lung cancer cells with ABCE1 siRNA and 5-FU exhibited synergistic or additive effects; however, ABCE1 siRNA and irinotecan had unexpected antagonistic effects. Our findings demonstrate that the strategy of downregulation of ABCE1 may be included in conventional 5-FU chemotherapy for lung cancer, minimizing the usage of 5-FU at high dosages.Hacettepe University, Scientific Research Projects Coordination UnitHacettepe University [968]This work was financially supported by Hacettepe University, Scientific Research Projects Coordination Unit (Grant No. 968)

The effect of calcium chloride concentration on alginate/Fmoc-diphenylalanine hydrogel networks

Bayram, Cem/0000-0001-8717-4668; CELIK, EKIN/0000-0003-1966-3907WOS: 000377737000027PubMed: 27207058Peptide based hydrogels gained a vast interest in the tissue engineering studies thanks to great superiorities such as biocompatibility, supramolecular organization without any need of additional crosslinker, injectability and tunable nature. Fmoc-diphenylalanine (FmocFF) is one of the earliest and widely used example of these small molecule gelators that have been utilized in biomedical studies. However, Fmoc-peptides are not feasible for long term use due to low stability and weak mechanical properties at neutral pH. In this study, Fmoc-FF dipeptides were mechanically enhanced by incorporation of alginate, a biocompatible and absorbable polysaccharide. The binary hydrogel is obtained via molecular self-assembly of FmocFF dipeptide in alginate solution followed by ionic crosslinldng of alginate moieties with varying concentrations of calcium chloride. Hydrogel characterization was evaluated in terms of morphology, viscoelastic moduli and diffusional phenomena and the structures were tested as 3D scaffolds for bovine chondrocytes. In vitro evaluation of scaffolds lasted up to 14 days and cell viability, sulphated glycosaminoglycan (sGAG) levels, collagen type II synthesis were determined. Our results showed that alginate incorporation into FmocFF hydrogels leads to better mechanical properties and higher stability with good biocompatibility. (C) 2016 Elsevier B.V. All rights reserved.Hacettepe University Scientific Research Projects Coordination Office (HU-BAP) [1136]This study was supported by the Hacettepe University Scientific Research Projects Coordination Office (HU-BAP, project no: 1136)

Calcified and mechanically debilitated three-dimensional hydrogel environment induces hypertrophic trend in chondrocytes

Bayram, Cem/0000-0001-8717-4668; CELIK, EKIN/0000-0003-1966-3907WOS: 000382858400005Currently, the main focus on tissue engineering strategies is to mimic the extracellular matrix of the related tissues. Many studies accomplished to build tissue scaffolds to act as the natural surroundings of the specific interest, which can be established to behave like either healthy or unhealthy tissues. The latter one of these conditions is a quite new approach and crucial for the design of three-dimensional in vitro disease models. This study investigates the potential of a composite scaffold consisting hydroxyapatite-integrated fluorenyl-9-methoxycarbonyl diphenylalanine hydrogels by focusing on the optimization of this hybrid scaffold for the development of an in vitro model of degenerative cartilage. Cell growth, chondrocyte proliferation, extracellular matrix production, hypertrophy marker monitoring, scaffold mechanical properties, and morphological analysis were evaluated. Fluorenyl-9-methoxycarbonyl diphenylalanine dipeptides were dissolved in null cell culture media and pH decreased sequentially to compel peptides to self-organize into fibrous hydrogel scaffolds. Nano-hydroxyapatite crystals were incorporated into fluorenyl-9-methoxycarbonyl diphenylalanine hydrogels during the gelation to investigate the effect on chondrocytes. It is observed that hydroxyapatite incorporation into peptide hydrogels significantly increased the alkaline phosphatase activity and assymetrical cell divisions, which is appraised as an outcome of chondrocyte hypertrophy. It is concluded that chondrocytes develop a hypertrophic potential when they are cultured in a media with nano-hydroxyapatites in a three-dimensional cell culture matrix mimicking the extracellular matrix conditions of degenerative cartilage

Designing siRNA-conjugated plant oil-based nanoparticles for gene silencing and cancer therapy

WOS: 000487149700001PubMed ID: 31509450TARAMASCOPUSTARAMAPUBMEDTARAMAWOSIn this study, the anticancer activities of two siRNA carriers were compared using a human lung adenocarcinoma epithelial cell line (A549). Firstly, poly(styrene)-graft-poly(linoleic acid) (PS-g-PLina) and poly(styrene)-graft-poly(linoleic acid)-graft-poly(ethylene glycol) (PS-g-PLina-g-PEG) graft copolymers were synthesized by free-radical polymerization. PS-PLina and PS-PLina-PEG nanoparticles (NPs) were prepared by solvent evaporation method and were then characterized. The size was found as 150 +/- 10 nm for PS-PLina and 184 +/- 6 nm for PS-PLina-PEG NPs. The NPs were functionalized with poly(l-lysine) (PLL) for c-myc siRNA conjugation. siRNA entrapment efficiencies were found in the range of 4-63% for PS-PLina-PLL and 6-42% for PS-PLina-PEG-PLL NPs. The short-term stability test was realised for 1 month. siRNA release profiles were also investigated. In vitro anticancer activity of siRNA-NPs was determined by MTT, flow cytometry, and fluorescence microscopy analyses. Obtained findings showed that both NPs systems were promising as siRNA delivery tool for lung cancer therapy.Bulent Ecevit University Research FundBulent Ecevit University [BEU-2016-33496813-01]; Kapadokya University [KUN(0).2018-BAGP-001]This work was financially supported by Bulent Ecevit University Research Fund (Grant no. BEU-2016-33496813-01) and Kapadokya University #KUN(0).2018-BAGP-001

Therapeutic potential of inhibiting ABCE1 and eRF3 genes via siRNA strategy using chitosan nanoparticles in breast cancer cells

ASIK, MEHMET/0000-0001-9154-2697; asik, Mehmet/0000-0001-9154-2697; Bayram, Cem/0000-0001-8717-4668WOS: 000355129700005In recent years, targeted cancer therapy strategies have begun to take the place of the conventional treatments. Inhibition of the specific genes, involved in cancer progress, via small interfering RNA (siRNA) has become one of the promising therapeutic approaches for cancer therapy. However, due to rapid nuclease degradation and poor cellular uptake of siRNA, a suitable carrier for siRNA penetration inside the cells is required. We used chitosan nanoparticles (CS-NPs) to efficiently deliver ATP-binding casette E1 (ABCE1) and eukaryotic release factor 3 (eRF3)-targeting siRNAs, individually and together, to reduce the proliferation and induce the apoptosis of breast cancer cells. The CS-NPs were generated by ionic gelation method using tripolyphosphate (TPP) as a crosslinker. Nanoparticles (NPs) were obtained with diameters ranging between 110 and 230 nm and the zeta potential of approximately 27 mV optimizing the solution pH to 4.5 and CS/TPP mass ratio to 3: 1. Loading efficiencies of 98.69 % +/- 0.051 and 98.83 % +/- 0.047 were achieved when ABCE1 siRNA and eRF3 siRNA were entrapped into the NPs, respectively. Cell proliferation assay demonstrated that siRNA-loaded CS-NPs were more effective on cancer cells when compared to siRNAs without CS-NPs. Parallel results were also obtained by apoptosis/necrosis, double-staining analysis. Within our study, the potency of ABCE1 and eRF3 siRNAs were shown for the first time with this kind of polymeric delivery system. The results also indicated that ABCE1 and eRF3, important molecules in protein synthesis, could serve as effective targets to inhibit the cancer cells.Hacettepe University, Scientific Research Projects Coordination UnitHacettepe University [011A601003]Our study was financially supported by Hacettepe University, Scientific Research Projects Coordination Unit (Grant number 011A601003)