Search

This review aims to highlight the use of RAFT polymerization in the synthesis of polymer bioconjugates. It covers two main bioconjugation strategies using the RAFT process: (i) post-polymerization bioconjugations using pre-synthesized reactive polymers, and (ii) bioconjugations via in situ polymerization using biomolecule-modified monomers or chain transfer agents. © 2011 The Royal Society of Chemistry.

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2019, Includes bibliographical references (leaves: 40-48), Text in English; Abstract: Turkish and English, Pathogens present in the food we consume and the water we drink pose a major threat to human health. Another major health concern is the metastasis of cancer in which cancer cells spread to new areas of the body, often by way of the lymph system or bloodstream. To minimize the burden on health and economy, the detection of biomicroparticles such as pathogens or circulating cancer cells in a highly sensitive and practical manner is higly desirable. This thesis aims to develop a method to create graphene-based biosensor substrate for detection of biomicroparticles such as bacteria, viruses or mammalian cells. For this aim, graphene surface was first functionalized using a linker molecule. The effect of solvent type on functionalization was investigated via Raman spectroscopy and X-Ray spectroscopy (XPS). AntiCD2 antibodies (Ab), as a model antibody, were then conjugated to the functionalized graphene via NHS/EDC chemistry. The Ab conjugation was verified by Raman spectroscopy and XPS analyses. Finally, Jurkat cells, as model biomicroparticles, were recognized and captured by Abconjugated graphene surface, as evidenced by optical microscopy. The temperature, medium, and method for interaction of cells with graphene surfaces as well as the specificity of the Ab- functionalized graphene surface were investigated. The results overall showed the specific and efficient recognition of model cell line by Abconjugated graphene surfaces., Tükettiğimiz yiyeceklerde ve içtiğimiz sularda bulunan patojenler insan sağlığı için büyük bir tehdit oluşturmaktadır. Bir diğer önemli sağlık sorunu, kanser hücrelerinin genellikle lenf sistemi veya kan dolaşımı yoluyla vücudun yeni bölgelerine yayıldığı kanser metastazıdır. Sağlık ve ekonomi üzerindeki yükü en aza indirmek için, patojenler veya dolaşımdaki kanser hücreleri gibi biyomikropartiküllerin oldukça hassas ve pratik bir şekilde saptanması talep edilen bir durumdur. Bu tez, bakteri, virüs veya memeli hücresi gibi biyomikropartiküllerin tespiti için grafen esaslı biyosensör substratı oluşturmak için bir yöntem geliştirmeyi amaçlamaktadır. Bu amaçla grafen yüzey ilk olarak bir bağlayıcı ara molekül kullanılarak fonksiyonelleştirildi. Çözücü tipinin grafen yüzey fonksiyonelleştirilmesi üzerindeki etkisi Raman spektroskopisi ve X-Ray spektroskopisi (XPS) ile araştırıldı. Daha sonra bir model antikor olarak AntiCD2 antikorları (Ab), NHS / EDC kimyası yoluyla fonksiyonelleştirilmiş grafen yüzeye konjüge edildi. Ab konjugasyonu Raman spektroskopisi ve XPS analizleriyle tespit edilmiştir. Son olarak, model biyomikropartiküller olarak kullanılan Jurkat hücreleri, Ab ile konjuge grafen yüzeyi tarafından tanındı ve hücre-yüzey etkileşimi optik mikroskopi ile kanıtlandı. Hücrelerin grafen yüzeyleri ile etkileşimi için sıcaklık, ortam ve yöntem ile Abfonksiyonel grafen yüzeyinin özgüllüğü araştırıldı. Sonuçlar genel olarak Abkonjuge grafen yüzeyler tarafından model hücre hattının spesifik ve etkili bir şekilde tanınmasını gösterdi., TUBITAK (117F186)

Thesis (Master)--Izmir Institute of Technology, Biotechnology and Bioengineering, Izmir, 2019, Includes bibliographical references (leaves. 57-61)., Text in English; Abstract: Turkish and English, Silk consists of two main proteins called fibroin and sericin. While fibroin is used in textile production and various biomaterial applications, sericin is considered as waste material in the textile industry. Sericin is a multi-component protein with an indefinite structure and it has been shown to be biocompatible and has biological activity. Because of the positive effects on keratinocytes and fibroblasts have led to the development of sericin-based biomaterials for the repair of skin tissue. Sericin from silkworm cocoons can be obtained by chemical treatment, enzymatic treatment and boiling in water. Although sericin can be separated from fibroin by chemical, enzymatic and boiling in water treatment methods, all these treatment methods are not enough to obtain recovery of high-quality sericin. Moreover, in these treatment methods, the exposure of sericin protein to high temperature causes even sericin protein obtained by the same method to indicate different characteristics. The fact that the obtained sericin demonstrate such major changes in the structure according to treatment methods bring inconsistencies in the quality of sericin produced as a biomaterial. The aim of the study is to produce native sequence of sericin that forms a tetramer contain each containing 38 amino acids with recombinant production in E.coli and to characterize structural properties Thus, obtaining sericin protein from the bacteria with recombinant methods will solve these problems in question The results indicate that for the first time, the conformational properties of recombinant sericin were obtained similar to the native sericin structure., Fibroin ile birlikte ipeği oluşturan proteinden biri olan serisin, fibroinlerin birbirine bağlanmasına yardımcı olarak kozanın oluşmasını sağlar. Fibroin tekstil üretiminde ve çeşitli biyomalzeme uygulamaların da kullanılırken, serisin tekstil endüstrisinde bir atık malzeme olarak kabul edilmektedir. Serisin proteinin biyolojik aktiviteye sahip olduğu ve biyouyumlu olduğu yapılan çalışmalarla gösterilmiştir. Serisin değişken aminoasit bileşimi ve çeşitli fonksiyonel grupları ile biyomedikal uygulamalar için ilgi çekici biyoaktif özelliklere sahiptir. Antioksidan karakteri ve memeli hücreleri üzerindeki mitojenik etkisi nedeniyle serisinin hücre kültürü ve doku mühendisliğinde yararlı olduğu son yıllarda yapılan çalışmalarla gösterilmiştir. Ayrıca, keratinositler ve fibroblastlar üzerindeki olumlu etkileri, başta yara bakım malzemeleri olmak üzere deri dokusu onarımı için serisin bazlı biyomalzemelerin gelişmesine yol açmıştır. İpek böceği kozalarından serisin kimyasal muamele, enzimatik muamele ve suda kaynatma yöntemleri ile elde edilebilir. Kimyasal ve enzimatik muamele yöntemleriyle ile serisin fibroinden ayrılabilse de bu yöntemlerle serisin elde edilirken protein yüksek sıcaklıklara maruz kaldığından tutarlı ve özellikleri öngörülebilen serisin elde edilememektedir. Farklı zamanlarda üretilen serisin farklı kalitede ve miktarda elde edilmektedir. Elde edilen serisinin ayrıştırma yöntemine göre yapısında büyük değişiklikler göstermesi, serisinden elde edilen biyomalzemelerin kalitesinde tutarsızlıklar meydana getirmektedir. Çalışmanın amacı, doğal serisin sekansını 4 kez tekrar eden 38 amino asit dizisinden oluşan tetrameri E. coli’de rekombinant yöntem ile üretmek ve yapısal özelliklerini karakterize etmektir. Böylelikle, bakterilerden rekombinant yöntemlerle serisin proteini elde edilmesi, söz konusu sorunları çözecektir. Sonuçlar, ilk kez rekombinant yöntem ile üretilen serisin proteinin konformasyon özelliklerinin, doğal serisin yapısına benzer şekilde elde edildiğini gösterdi., TUBITAK (117Z841)

Thesis (Doctoral)--Izmir Institute of Technology, Materials Science and Engineering, Izmir, 2019, Includes bibliographical references (leaves: 105-118), Text in English; Abstract: Turkish and English, The aim of this thesis is to synthesize gold nanorods (AuNRs) and lipid-stabilized nanobubbles containing AuNRs and investigate the potential of these plasmonic nanostructures as photothermal therapy agents for breast and prostate cancer through in vitro cell culture experiments. For this aim, firstly, AuNRs were synthesized at varying aspect ratios (ARs) and characterized via several techniques including UV-Vis/NIR spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), inductively coupled plasma-mass spectroscopy (ICP-MS), electrophoretic light scattering (ELS) and X-ray photoelectron spectroscopy (XPS). The surface of AuNRs was modified with a biocompatible polymer, poly(ethylene glycol) (PEG), via ligand exchange method. Cytotoxicity, cell uptake and photothermal effects of AuNRs were investigated via in vitro cell culture experiments using human prostate cancer (DU 145) and epithelial (RWPE-1), breast cancer (MCF7) and epithelial (MCF 10A) cell lines. It was concluded that AuNRs (AR=4.0) were superior than AuNRs (AR=7.0) in terms of cell viability and photothermal effect. Separately, a non-commercial antibody (Ab) targeting a specific sialic acid derivative on the plasma membrane of DU 145 and MCF7 cancer cells was conjugated to AuNRs. Conjugations were characterized with the same techniques and investigated via in vitro cytotoxicity and cell uptake experiments. The Ab-conjugated AuNRs displayed the capability of selective targeting prostate cancer cells. Additionally, lipid-stabilized AuNRs and lipid-stabilized nanobubbles containing AuNRs (AuNBs) were synthesized for the first time and characterized using UV-Vis/NIR spectroscopy, SEM, ICP-MS and ELS techniques. Lipid-stabilized AuNRs were successfully synthesized using varying lipid mixtures instead of cationic, toxic surfactant. Separately, AuNBs were synthesized by combining PEG modified AuNRs with DPPC: DSPE-PEG lipid film under sonication and gas stream. AuNBs showed the same or significantly lower toxicity depending on the cell types and the same photothermal effect with respect to AuNRs (AR=4.0) upon irradiation under laser at 808 nm., Bu tezin amacı, altın nanoçubukları (AuNRs) ve altın nanoçubukları içeren lipit stabilize edilmiş nanobalonları sentezlemek ve bu plazmonik nanoyapıların, in vitro hücre kültürü deneyleri aracılığıyla meme ve prostat kanseri için fototermal terapi maddeleri olarak potansiyelini araştırmaktır. Bu amaçla, ilk olarak, altın nanoçubuklar değişen boy-en oranlarında sentezlendi ve UV-Vis/NIR spektroskopisi, taramalı elektron mikroskobu (SEM), atomik kuvvet mikroskobu (AFM), indüktif olarak eşleşmiş plazma kütle spektroskopisi (ICP-MS), elektroforetik ışık saçılımı (ELS) ve X-ışını fotoelektron spektroskopisi (XPS) dahil olmak üzere çeşitli tekniklerle karakterize edildi. Altın nanoçubukların yüzeyi, ligand değişim metodu ile biyouyumlu bir polimer olan poli(etilen glikol) (PEG) ile modifiye edildi. Altın nanoçubukların sitotoksisitesi, hücre alımı ve fototermal etkileri, insan prostat kanser (DU 145) ve epitel (RWPE-1), meme kanser (MCF7) ve epitel (MCF 10A) hücre hatları kullanılarak in vitro hücre kültürü deneyleri ile araştırıldı. Hücre canlılığı ve fototermal etki açısından boy-en oranı 4 olan altın nanoçubukların boy-en oranı 7 olandan daha üstün olduğu sonucuna varıldı. Ayrı olarak, DU 145 ve MCF7 kanser hücrelerinin plazma zarı üzerinde spesifik bir sialik asit türevini hedef alan bir antikor, altın nanoçubuklara konjuge edildi. Konjugasyonlar aynı teknikler ile karakterize edildi ve in vitro sitotoksisite ve hücre alımı deneyleri ile araştırıldı. Antikor ile konjuge altın nanoçubuklar prostat kanseri hücrelerini seçici hedefleme yeteneği göstermiştir. Ek olarak, lipit stabilize edilmiş altın nanoçubuklar ve altın nanoçubukları içeren lipit stabilize edilmiş nanobalonlar ilk kez sentezlendi ve UV-Vis/NIR spektroskopisi, SEM, ICP-MS ve ELS yöntemleri kullanılarak karakterize edildi. Katyonik, toksik yüzey aktif madde yerine çeşitli lipit karışımları kullanılarak lipit stabilize altın nanoçubuklar başarıyla sentezlendi. Ayrıca, altın nanobalonlar sonikasyon ve gaz akışı altında PEG modifiye altın nanoçubukların DPPC: DSPE-PEG lipit filmi ile birleştirilmesiyle sentezlendi. Altın nanobalonlar boy-en oranı 4 olan altın nanoçubuklara göre, hücre tiplerine bağlı olarak önemli ölçüde daha düşük veya aynı toksisite ve 808 nm lazer ışıması altında aynı fototermal etki gösterdi., TUBITAK/TBAG/112T507, TUBITAK/MAG/213M673, TUBITAK/MFAG/117F186

Bulmus, Volga/0000-0001-9944-1444, WOS: 000434745200044, Synthetic peptides with cyclic arginine-glycine-aspartate motif (cRGD) play an important role in cell recognition and cell adhesion. cRGD-decorated soluble polymers and polymeric nanoparticles have been increasingly used for cell-specific delivery of antitumor drugs. While the significance of cRGD modification for tumor cell-specific targeting of polymeric carriers is well-accepted, straightforward procedures ensuring the fidelity of cRGD modification of polymeric systems are still lacking. Herein, we have reported an in-situ polymerization approach for synthesis of cRGD-end-functionalized well-defined polymers as potential building blocks of targeted drug delivery systems. A new cRGD peptide functionalized RAFT agent was synthesized as confirmed by MALDI-TOF and H-1 NMR spectroscopy. The ability of this RAFT agent to control polymerizations was then tested using two different monomers oligoethyleneglycol acrylate and t-butyl methacrylate. The RAFT-controlled character of polymerizations and the living characteristic of the synthesized polymers were investigated through a series of kinetic experiments. The cytotoxicity and targeting capability of cRGD-functionalized OEGA polymers were investigated using cell lines expressing alpha(v)beta(3) integrins at varying extents.

PEGylation, covalent attachment of PEG to therapeutic biomolecules, in which suboptimal pharmacokinetic profiles limiting their therapeutic utility are of concern, is a widely applied technology. However, this technology has been challenged by reduced bioactivity of biomolecules upon PEGylation and immunogenicity of PEG triggering immune response and abrogating clinical efficacy, which collectively necessitate development of stealth polymer alternatives. Here we demonstrate that comb-shape poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), a stealth polymer alternative, has a more compact structure than PEG and self-organize into nanoparticles in a molecular weight dependent manner. Most notably, we show that comb-shape POEGMA promotes significantly higher cellular uptake and exhibits less steric hindrance imposed on the conjugated biomolecule than PEG. Collectively, comb-shape POEGMA offers a versatile alternative to PEG for stealth polymer-biomolecule conjugation applications., Scientific and Technological Research Council of Turkey (TUBITAK 113Z823)

Diblock copolymers of 4-vinylpyridine (4VP) and oligoethyleneglycol methyl ether methacrylate (OEGMA) were synthesized for the first time using RAFT polymerization technique as potential drug delivery systems. Effects of the number of ethylene glycol units in OEGMA, chain length of hydrophobic P4VP block, pH, concentration and temperature on the solution behavior of the copolymers were investigated comprehensively. Copolymer chains formed micelles at pH values higher than 5 whereas unimeric polymers were observed to exist below pH 5, owing to the repulsion between positively charged P4VP blocks. The size of the micelles was dependent on the relative length of blocks, P4VP and POEGMA. Thermo-responsive properties of copolymers were investigated depending on the pH and length of P4VP block. The increase in the length of P4VP block decreased the LCST substantially at pH 7. At pH 3, LCST of copolymers shifted to higher temperatures due to the increased interaction of copolymers with water through positively charged P4VP block., The Scientific and Technological Research Council of Turkey (110T570)

In this study, interactions of polyethylene glycol (PEG)-coated gold nanoparticles (AuNPs) with cells were investigated with particular focus on the relationship between the PEG layer properties (conformation, grafting density, and hydrodynamic volume) and cell cycle arrest, apoptosis, and DNA damage. Steric hindrance and PEG hydrodynamic volume controlled the protein adsorption, whereas the AuNP core size and PEG hydrodynamic volume were primary factors for cell uptake and viability. At all PEG grafting densities, the particles caused significant cell cycle arrest and DNA damage against CaCo2 and PC3 cells without apoptosis. However, at a particular PEG grafting density (∼0.65 chains/nm2), none of these severe damages were observed on 3T3 cells indicating discriminating behavior of the healthy (3T3) and cancer (PC3 and CaCo2) cells. It was concluded that the PEG grafting density and hydrodynamic volume, tuned with the PEG concentration and AuNP size, played an important role in particle-cell interactions., Izmir Institute of Technology (2011IYTE10)

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., The Scientific and Technological Research Council of Turkey (111T960)

Surface modification with linear polymethacrylic acid (20 kDa), linear and branched polyethylenimine (25 kDa), and branched oligoethylenimine (800 Da) is commonly used to improve the function of magnetite nanoparticles (MNPs) in many biomedical applications. These polymers were shown herein to have different adsorption capacity and anticipated conformations on the surface of MNPs due to differences in their functional groups, architectures, and molecular weight. This in turn affects the interaction of MNPs surfaces with biological serum proteins (fetal bovine serum). MNPs coated with 25 kDa branched polyethylenimine were found to attract the highest amount of serum protein while MNPs coated with 20 kDa linear polymethacrylic acid adsorbed the least. The type and amount of protein adsorbed, and the surface conformation of the polymer was shown to affect the size stability of the MNPs in a model biological media (RPMI-1640). A moderate reduction in r 2 relaxivity was also observed for MNPs suspended in RPMI-1640 containing serum protein compared to the same particles suspended in water. However, the relaxivities following protein adsorption are still relatively high making the use of these polymer-coated MNPs as Magnetic Resonance Imaging (MRI) contrast agents feasible. This work shows that through judicious selection of functionalization polymers and elucidation of the factors governing the stabilization mechanism, the design of nanoparticles for applications in biologically relevant conditions can be improved. © 2012 American Chemical Society., ARC (DP0985848); Spanish ISCIII-MSPS (CD09/00030)

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., NHMRC Senior Research Fellowship (APP1058299)

A spermine-like polymer was synthesized via reversible addition- fragmentation chain transfer polymerization as a potential endosomal escaping agent. A new methacrylate monomer, 2-((tert-butoxycarbonyl)(2-((tert- butoxycarbonyl)amino)ethyl)amino)ethylmethacrylate (BocAEAEMA), was prepared and then polymerized via RAFT polymerization at constant monomer or initiator concentration at varying [M]/[R]/[I] ratios. In all polymerizations, ln[M] 0/[M] increased linearly with time. The linear increase in M n with monomer conversion was also observed. P(BocAEAEMA)s with controlled molecular weights and narrow molecular weight distributions were obtained. The in vitro cytotoxicity and proton sponge capacity of deprotected polymers P(AEAEMA) were investigated in comparison with a widely used endosomal-disruptive polymer, PEI. P(AEAEMA)s were found to possess proton sponge capacity comparable with PEI. More importantly, P(AEAEMA)s were not toxic on NIH 3T3 cells at concentrations where PEI (25 kDa) was highly toxic (0.4 μM and above). P(AEAEMA) was able to fully condense a DNA fragment at nitrogen/phosphate (N/P) ratios of 10 and above, as evidenced by gel electrophoresis. P(BocAEAEMA) was then chain-extended with a model sugar monomer, mannose-acrylate (ManAc), to yield P(AEAEMA)-b-P(ManAc) block copolymers, to potentially provide cell-recognition ability to the polyplex particles. Although the presence of the P(ManAc) block partially inhibited the interaction of P(AEAEMA) with DNA, P(AEAEMA)13-b-P(ManAc)7 was able to form polyplexes with DNA at N/P ratios ranging between 20/1 and 2/1. Dynamic light scattering measurements showed that while P(AEAEMA) (M n = 5.5 kDa) and DNA formed polyplex particles having a hydrodynamic diameter (Dh) of 125 ± 51 nm, P(AEAEMA)13-b- P(ManAc)7 and DNA formed particles with a smaller Dh of 38 ± 10 nm., UK-Turkey Higher Education Knowledge Partnership Programme (TR1012012/KP25); Bioengineering Research and Application Centre (Izmir Institute of Technology, Turkey); Izmir Institute of Technology (BAP2011lYTE04)

Dissolved air flotation (DAF), an effective treatment method for clarifying algae/cyanobacteria-laden water, is highly dependent on coagulation-flocculation. Treatment of algae can be problematic due to unpredictable coagulant demand during blooms. To eliminate the need for coagulation-flocculation, the use of commercial polymers or surfactants to alter bubble charge in DAF has shown potential, termed the PosiDAF process. When using surfactants, poor removal was obtained but good bubble adherence was observed. Conversely, when using polymers, effective cell removal was obtained, attributed to polymer bridging, but polymers did not adhere well to the bubble surface, resulting in a cationic clarified effluent that was indicative of high polymer concentrations. In order to combine the attributes of both polymers (bridging ability) and surfactants (hydrophobicity), in this study, a commercially-available cationic polymer, poly(dimethylaminoethyl methacrylate) (polyDMAEMA), was functionalised with hydrophobic pendant groups of various carbon chain lengths to improve adherence of polymer to a bubble surface. Its performance in PosiDAF was contrasted against commercially-available poly(diallyl dimethyl ammonium chloride) (polyDADMAC). All synthesised polymers used for bubble surface modification were found to produce positively charged bubbles. When applying these cationic micro-bubbles in PosiDAF, in the absence of coagulation-flocculation, cell removals in excess of 90% were obtained, reaching a maximum of 99% cell removal and thus demonstrating process viability. Of the synthesised polymers, the polymer containing the largest hydrophobic functionality resulted in highly anionic treated effluent, suggesting stronger adherence of polymers to bubble surfaces and reduced residual polymer concentrations., Australian Research Councils Linkage Project (LP0990189); SA Water; Veolia Water; United Water; Melbourne Water; Seqwater; Australian Research Council Post Doctoral Industry Fellowship (LP0990189); Australian Research Council Postgraduate Award Industry scholarship; Water Research Australia (4025-10)

Bu tezin ilk bölümünde, görüntüleme veya tanı amaçlı tasarlanmış polietilen glikol (PEG) modifiye altın nanotaneciklerin, boyut, PEG tabakası yoğunluğu ve konformasyon gibi özelliklerinin, hücre alımı, toksisite ve hücre döngüsü fazları üzerindeki etkileri Prostat (PC3), kolon (CaCo2) kanser hücre hatlarında ve 3T3 İsviçre fibroblast hücrelerine karşı araştırılmıştır. PEG kaplı taneciklerin hücre alımı ve toksisite profillerinin, boyut, yüzey özellikleri ve hücre tipine bağlı olarak değiştiği görülmüştür. Taneciklerin belirli dozlarda apoptotik davranış olmadan DNA hasarına neden olarak hücre döngüsü fazlarında değişikliğe neden olduğu saptanmıştır. Bu tezin ikinci bölümünde, altın nanotanecik (AuNPs), katyonik pentablok kopolimer veya füzyojenik peptit bazlı, kırılabilir disulfit bağları ve elektrostatik etkileşimler ile elde edilen cok katmanlı siRNA tasinim sistemleri geliştirilmiştir. siRNA ve katyonik pentablok kopolimer veya peptit arasındaki doğrudan elektrostatik etkileşimler sonucu elde edilen siRNA/polimer (polipleks) ve siRNA/peptit (peptitpleks) kompleksleri, sırasıyla, kontrol olarak kullanılmıştır. Buna ek olarak, siRNA ve füzyojenik peptit arasında kırılabilir disülfit bağları kullanılarak elde edilen konjuge sistem alternatif bir siRNA taşınım sistemi olarak önerilmiştir. Geliştirilen sistemlerin siRNA aktivitesi, toksisite, hücre alımı ve hücre içi dağılımı gibi özellikleri lusiferaz salgılayan SKOV3 yumurtalık kanseri hücre hattına karşı incelenmiştir. Altın nanotanecik bazlı çok katmanlı sistemlerde ve komplex sistemlerde katyonik pentablok kopolimerlerin veya füzyojenik peptitlerin kullanılması, etkili siRNA yüklemesi ve nükleaz enzimleri ve serum proteinlerinden korunma sağlamasına ek olarak, etkin bir hücre alımı, endozomal kaçış ve sitoplazmada siRNA aktivitesi sağlamıştır. Altın nanotanecik bazlı çok katmanlı sistemlerde kırılabilir disülfit bağlarının mevcudiyeti, bu sistemlerin aynı miktarda siRNA içeren polipleks ve peptitpleks sistemlere oranla herhangi bir toksik etki göstermeden ~% 20 oranda daha etkili bir siRNA aktivitesi gösterdiği saptanmıştır. In the first part of this thesis, a comprehensive characterization of polyethylene glycol (PEG) modified AuNPs designed for imaging or diagnostic purposed was carried out to investigate the effect of the size, PEG layer conformation and grafting density on the cellular uptake, toxicity and cell cycle phases against prostate (PC3), colon (CaCo2) cancer cell lines and 3T3 Swiss fibroblast cells. It was noticed that the cellular uptake and toxicity profiles of the particles varied depending on the size, surface properties and cell type. The particles were found to show alterations in cell cycle phases by causing DNA damage without apoptotic behavior at certain doses. In the second part of this thesis, efficient multilayer small interfering RNA (siRNA) delivery systems based on gold nanoparticles (AuNPs), cationic pentablock copolymers or fusogenic peptides were developed using cleavable disulfide bonds and electrostatic interactions. siRNA/Polymer (polyplexes) and siRNA/Peptide (peptideplexes) complexes formed by direct electrostatic complexation between siRNA and the cationic pentablock copolymers or peptides were used as controls, respectively. In addition, a conjugate siRNA delivery system based on the cleavable disulfide bonds between siRNA and fusogenic peptide was also proposed as an alternative system. The siRNA activity, toxicity, cellular uptake and intracellular distribution of the developed systems were investigated against luciferase-expressing SKOV3 ovarian cancer cell line. The use of cationic block copolymers or fusogenic peptides in AuNP based multilayer systems and complex systems, provided efficient siRNA condensation and protection from nuclease enzyme and serum protein degradation, in addition to cellular uptake, endosomal escape and siRNA activity in the cytoplasm. 172

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., Australian Research Council (ARC)

Well-defined amphiphilic block copolymers composed of hydrophilic and hydrophobic blocks linked through an acid-labile acetal bond were synthesized directly by RAFT polymerization using a new poly(ethylene glycol) (PEG) macroRAFT agent modified with an acid-labile group at its R-terminal. The new macroRAFT agent was used for polymerization of poly(t-butyl methacrylate) (PtBMA) or poly(cholesterol-methacrylate) (PCMA) to synthesize well-defined block copolymers with a PEG block sheddable under acidic conditions. The chain extension polymerization kinetics showed known traits of RAFT polymerization. The molecular weight distributions of the copolymers prepared using the new macroRAFT agent remained below 1.2 during the polymerizations and the molecular weight of the copolymers was linearly proportional to monomer conversions. The acid-catalyzed hydrolysis behavior of the PEG-macroRAFT agent and the PEG-b-PtBMA (Mn = 13,600 by GPC, PDI = 1.10) was studied by GPC, 1H NMR and UV-vis spectroscopy. The half-life of acid-hydrolysis was 70 min at pH 2.2 and 92 h at pH 4.0. The potential use of the pH-labile shedding behavior of the copolymers was demonstrated by conjugating a thiol-modified siRNA to ω-pyridyldisulfide modified PEG-b-PCMA. The resultant PEG-b-PCMA-b-siRNA triblock modular polymer released PCMA-b-siRNA segment in acidic and siRNA segment in reductive conditions, as confirmed by polyacrylamide gel electrophoresis., Australian Research Council (ARC) (DP 0770818)

Poly(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., Australian Research Council (ARC) (DP 0770818)

11th IEEE International Conference on Nanotechnology, NANO 2011; Portland, OR; United States; 15 August 2011 through 19 August 2011, Size stability of magnetite nanoparticles (MNP) with different surface functional groups in biological media was achieved by the addition of fetal bovine serum (FBS). The stability of the particles was attributed to the formation of protein coronas around the particles, which provides sufficient steric hindrance to prevent aggregation of the particles. The stability of different modified MNP were also studied in biological media containing bovine serum albumin (BSA) to further understand the stabilization mechanism. BSA was found to stabilize polyethyleneimine (PEI) modified MNP and polymethacrylic acid (PMAA) coated MNP, but not the bare MNP. These results indicate a difference in interactions between serum protein and the MNP that is govern by the type of functional groups on the MNP surface, with positively charged surface groups resulting higher protein adsorption and better stability. © 2011 IEEE., ARC Linkage program (LP0882720)

A 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%).