Chemical Engineering / Kimya Mühendisliği
Permanent URI for this collectionhttps://hdl.handle.net/11147/14
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Article Citation - WoS: 31Citation - Scopus: 32Synthesis, Self-Assembly and Stimuli Responsive Properties of Cholesterol Conjugated Polymers(Royal Society of Chemistry, 2012) Sevimli, Sema; Sagnella, Sharon; Kavallaris, Maria; Bulmuş, Volga; Davis, Thomas P.Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to generate well-defined pH-responsive biofunctional polymers as potential 'smart' gene delivery systems. A series of five poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) P(DMAEMA-co-CMA) statistical copolymers, with similar molecular weights and varying cholesterol content, were prepared. The syntheses, compositions and molecular weight distributions for P(DMAEMA-co-CMA) were monitored by nuclear magnetic resonance (NMR), solid-state NMR and gel permeation chromatography (GPC) evidencing well-defined polymeric structures with narrow polydispersities. Aqueous solution properties of the copolymers were investigated using turbidimetry and light scattering to determine hydrodynamic diameters and zeta potentials associated with the phase transition behaviour of P(DMAEMA-co-CMA) copolymers. UV-Visible spectroscopy was used to investigate the pH-responsive behaviour of copolymers. Hydrodynamic radii were measured in the range 10-30 nm (pH, temperature dependent) by dynamic light scattering (DLS). Charge studies indicated that P(DMAEMA-co-CMA) polymers have an overall cationic charge, mediated by pH. Potentiometric studies revealed that the buffering capacity and pK a values of polymers were dependent on cholesterol content as well as on cationic charge. The buffering capacity increased with increasing charge ratio, overall demonstrating transitions in the pH endosomal region for all five copolymeric structures. Cell viability assay showed that the copolymers displayed increasing cytotoxicity with decreasing number of cholesterol moieties. These preliminary results show the potential of these well-defined P(DMAEMA-co-CMA) polymers as in vitro siRNA delivery agents.Article Citation - WoS: 22Citation - Scopus: 25The Endocytic Pathway and Therapeutic Efficiency of Doxorubicin Conjugated Cholesterol-Derived Polymers(Royal Society of Chemistry, 2015) Sevimli, Sema; Sagnella, Sharon; Macmillan, Alexander; Whan, Renee; Kavallaris, Maria; Bulmuş, Volga; Davis, Thomas P.Previously synthesized poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA) copolymers were examined as potential drug delivery vehicles. P(MAA-co-CMA) copolymers were fluorescently labelled and imaged in SHEP and HepG2 cells. To understand their cell internalization pathway endocytic inhibition studies were conducted. It was concluded that P(MAA-co-CMA) are taken up by the cells via clathrin-independent endocytosis (CIE) (both caveolae mediated and cholesterol dependent endocytosis) mechanisms. The formation and characterization of P(MAA-co-CMA)-doxorubicin (DOX) nanocomplexes was investigated by fluorescence lifetime imaging microscopy (FLIM), UV-Visible spectroscopy (UV-Vis) and dynamic light scattering (DLS) studies. The toxicity screening between P(MAA-co-CMA)-DOX nanocomplexes (at varying w/w ratios) and free DOX, revealed nanocomplexes to exhibit higher cytotoxicity towards cancer cells in comparison to normal cells. FLIM and confocal microscopy were employed for investigating the time-dependent release of DOX in SHEP cells and the cellular uptake profile of P(MAA-co-CMA)-DOX nanocomplexes in cancer and normal cell lines, respectively. The endocytic pathway of P(MAA-co-CMA)-DOX nanocomplexes were examined in SHEP and HepG2 cells via flow cytometry revealing the complexes to be internalized through both clathrin-dependent (CDE) and CIE mechanisms. The drug delivery profile, reported herein, illuminates the specific endocytic route and therapeutic efficiency of P(MAA-co-CMA)-DOX nanocomplexes strongly suggesting these particles to be promising candidates for in vivo applications.Article Citation - WoS: 7Citation - Scopus: 7Assessment of Cholesterol-Derived Ionic Copolymers as Potential Vectors for Gene Delivery(American Chemical Society, 2013) Sevimli, Sema; Sagnella, Sharon; Kavallaris, Maria; Bulmuş, Volga; Davis, Thomas P.A library of cholesterol-derived ionic copolymers were previously synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization as 'smart' gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly(methacrylic acid-co-cholesteryl methacrylate) (P(MAA-co-CMA)) and cationic poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P(DMAEMA-co-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P(DMAEMA-co-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P(MAA-co-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P(MAA-co-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P(DMAEMA-co-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P(MAA-co-CMA)-DNA) and 20:1 (Q-P(DMAEMA-co-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P(MAA-co-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P(DMAEMA-co-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P(DMAEMA-co-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P(DMAEMA-co-CMA)-siRNA particles are extremely promising candidates for in vivo applications of siRNA therapy.Article Citation - WoS: 46Citation - Scopus: 46Conjugation of Sirna With Comb-Type Peg Enhances Serum Stability and Gene Silencing Efficiency(John Wiley and Sons Inc., 2011) Gunasekaran, Karthikeyan; Nguyen, Thi H.; Maynard, Heather D.; Davis, Thomas P.; Bulmuş, VolgaA thiol-modified siRNA targeting the enhanced green fluorescence protein (eGFP) gene was conjugated with RAFT-synthesized, pyridyl disulfide-functional poly(PEG methyl ether acrylate)s (p(PEGA)s). siRNA-p(PEGA) conjugates demonstrated significantly enhanced in vitro serum stability and nuclease resistance compared to the unmodified and thiol-modified siRNA. The complexes of siRNA-p(PEGA) conjugates with a fusogenic peptide, KALA ((+)/(-) = 2) inhibited the protein expression approximately 28-fold more than the KALA complex of the unmodified siRNA. The protein inhibition caused by siRNA-p(PEGA)-KALA complexes (56± 5%-58± 3% of the fluorescence expressed in non-treated cells) was comparable to the effect of the unmodified siRNA-lipofectamine complex (77± 7%).Article Citation - WoS: 24Citation - Scopus: 24Synthesis of Heterotelechelic Polymers With Affinity To Glutathione-S and Biotin-Tagged Proteins by Raft Polymerization and Thiol-Ene Reactions(Royal Society of Chemistry, 2011) Huang, Xin; Boyer, Cyrille; Davis, Thomas P.; Bulmuş, Volgaα-Glutathione (GSH), ω-biotin functionalized poly(N-isopropylacrylamide) (PNIPAAm) was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization using a new R-group allyl functionalized trithiocarbonate chain transfer agent (CTA) and thiol-ene reactions. GPC and 1H NMR results indicated that the allyl group had no adverse effect on the RAFT-controlled polymerization of NIPAAm and PEG-A, and the new CTA could efficiently control the polymerizations. Employing radical thiol-ene and Michael addition reactions, heterotelechelic α-allyl, ω-carboxylic acid-PNIPAAm was first aminolyzed in the presence of maleimide-modified biotin and subsequently reacted with GSH via radical thiol-ene addition to yield α-GSH, ω-biotin functionalized PNIPAAm. Glutathione S-transferase (GST) and streptavidin (SAv) were coupled in solution with heterofunctional PNIPAAm via bioaffinity interactions. Separately, α-GSH, ω-biotin functionalized PNIPAAm was further shown to bind GST-tagged Rac1, a potential cancer marker, and biotin-tagged bovine serum albumin (BSA).Article Citation - WoS: 18Citation - Scopus: 18Dicer-Labile Peg Conjugates for Sirna Delivery(American Chemical Society, 2011) Kow, Siew Ching; Mccorroll, Joshua A.; Valade, David; Boyer, Cyrille; Dwarte, Tanya; Davis, Thomas P.; Kavallaris, Maria; Bulmuş, VolgaPoly(ethylene glycol) (PEG) conjugates of Dicer-substrate small interfering RNA (DsiRNA) have been prepared to investigate a new siRNA release strategy. 3'-sense or 5'-antisense thiol-modified, blunt-ended DsiRNAs, inhibiting enhanced green fluorescent protein (eGFP) expression, were covalently conjugated to PEG with varying molecular weights (2, 10, and 20 kg/mol) through a stable thioether bond using a Michael addition reaction. The DsiRNA conjugates with 2 kg/mol PEG (both 3'-sense or 5'-antisense strand conjugated) and the 10 kg/mol PEG conjugated to the 3'-sense strand of DsiRNA were efficiently cleaved by recombinant human Dicer to 21-mer siRNA, as determined by gel electrophoresis. Importantly, 2 and 10 kg/mol PEG conjugated to the 3'-sense strand of DsiRNA showed potent gene silencing activity in human neuroblastoma (SH-EP) cells, stably expressing eGFP, at both the mRNA and protein levels. Moreover, the 10 kg/mol PEG conjugates of the 3'-sense strand of DsiRNA were less immunogenic when compared with the unmodified DsiRNA, determined via an immune stimulation assay on human peripheral blood mononuclear cells.