Browsing by Author "Sevimli, Sema"

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Citation - WoS: 7
Citation - Scopus: 7
Assessment of Cholesterol-Derived Ionic Copolymers as Potential Vectors for Gene Delivery
(American Chemical Society, 2013-11) Sevimli, Sema; Sagnella, Sharon; Kavallaris, Maria; Bulmuş, Volga; Davis, Thomas P.; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
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.
Citation - WoS: 22
Citation - Scopus: 25
The Endocytic Pathway and Therapeutic Efficiency of Doxorubicin Conjugated Cholesterol-Derived Polymers
(Royal Society of Chemistry, 2015-02) Sevimli, Sema; Sagnella, Sharon; Macmillan, Alexander; Whan, Renee; Kavallaris, Maria; Bulmuş, Volga; Davis, Thomas P.; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
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.
Raft-Synthesis of Cholesterol Conjugated Polymers as Potential Therapeutic Carriers
(American Chemical Society, 2011) Sevimli, Sema; İnci, Fatih; Bulmuş, Volga; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
[No abstract available]
Citation - WoS: 31
Citation - Scopus: 32
Synthesis, Self-Assembly and Stimuli Responsive Properties of Cholesterol Conjugated Polymers
(Royal Society of Chemistry, 2012-08) Sevimli, Sema; Sagnella, Sharon; Kavallaris, Maria; Bulmuş, Volga; Davis, Thomas P.; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
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.
Citation - WoS: 41
Citation - Scopus: 42
Well-Defined Cholesterol Polymers With Ph-Controlled Membrane Switching Activity
(American Chemical Society, 2012-10) Sevimli, Sema; İnci, Fatih; Zareie, Hadi M.; Bulmuş, Volga; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
Cholesterol has been used as an effective component of therapeutic delivery systems because of its ability to cross cellular membranes. Considering this, well-defined copolymers of methacrylic acid and cholesteryl methacrylate, poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA), were generated as potential delivery system components for pH-controlled intracellular delivery of therapeutics. Statistical copolymers with varying cholesterol contents (2, 4, and 8 mol %) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameters of the copolymers in aqueous solutions ranged from 5 ± 0.3 to 7 ± 0.4 nm for the copolymers having 2 and 4 mol % CMA and 8 ± 1.1 to 13 ± 1.9 nm for the copolymer having 8 mol % CMA with increasing pH (pH 4.5-7.4). Atomic force microscopy (AFM) analysis revealed that the copolymer having 8 mol % CMA formed supramolecular assemblies while the copolymers having 2 and 4 mol % CMA existed as unimers in aqueous solution. The pH-responsive behavior of the copolymers was investigated via UV-visible spectroscopy revealing phase transitions at pH 3.9 for 2 mol % CMA, pH 4.7 for 4 mol % CMA, and pH 5.4 for 8 mol % CMA. Lipid bilayers and liposomes as models for cellular membranes were generated to probe their interactions with the synthesized copolymers. The interactions were determined in a pH-dependent manner (at pH 5.0 and 7.4) using surface plasmon resonance (SPR) spectroscopy and liposome leakage assay. Both the SPR analyses and liposome leakage assays indicated that the copolymer containing 2 mol % CMA displayed the greatest polymer-lipid interactions at pH 5.0, presenting the highest binding ability to the lipid bilayer surfaces, and also demonstrating the highest membrane destabilization activity. CellTiter-Blue assay showed that the copolymers did not affect the cell viability up to 30 μM over a period of 72 h. © 2012 American Chemical Society.