Fiziksel ve kimyasal çapraz bağlı DNA hidrojellerinin sentezi ve karakterizasyonu
Synthesis and characterization of physical and chemical DNA hydrogels
Yazar: HÜSNİYE ÇAKMAK
Danışman: PROF. DR. OĞUZ OKAY
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı
Derece: Yüksek Lisans, 2013
Konu: Kimya = Chemistry ; Polimer Bilim ve Teknolojisi = Polymer Science and Technology
Dizin: DNA = DNA ; Hidrojeller = Hydrogels ; Jeller = Gels ; Polimer jel = Polymer gel ; Uzun etkili ilaçlar = Delayed action preparations

Özet:
Tüm yaşayan hücreler bünyelerinde baz diziliminde genetik bilgiler içeren DNA moleküllerini bulundururlar. Polimerik jeller sentetik polimerlerden elde edilebileceği gibi DNA molekülü gibi doğal polimerlerden de elde edilebilir. DNA hidrojelleri, çapraz bağlı DNA ipliklerinden oluşan ve sulu çözeltilerde şişmiş bir ağyapı olarak tanımlanabilir. Yüklü, yarı-esnek zincirlerden oluşan DNA hidrojellerinin kontrollü ilaç salımı, doku mühendisliği ve biyomedikal kullanımları gibi DNA'nın karakteristik özelliklerinden yararlanılan geniş uygulama alanları vardır. Bu çalışmanın amacı, dışarıdan gelen uyarılara hızlı cevap verebilen ve ilaç salımında kullanılmak üzere vücutta herhangi bir toksik etki bırakmayacak fiziksel çapraz bağlı DNA hidrojellerini sentezlemek ve karakterize etmektir. DNA temel hal durumunda bazları iki sarmala bağlayan hidrojen bağları sayesinde kararlı bir yapıya sahiptir ve bu konformasyon çift sarmal konformasyon olarak bilinir. Bu yapıdaki DNA'nın sulu çözeltisi ısıtıldığı zaman, sarmalları bir arada tutan bu hidrojen bağları kırılır ve çift sarmal yapı rastgele konformasyona sahip tek sarmallar haline parçalanır. Çift sarmal yapıdan tek sarmal yapıya geçiş erime veya denatürasyon olarak bilinmekte olup, bu proses seyreltik DNA çözeltilerinin yavaşça soğutulması durumunda tersinir olabilir. Fiziksel çapraz bağlı DNA hidrojelleri, NaBr çözeltisi içerisinde çözülen çifte sarmal yapıya sahip 2000 baz çifti içeren DNA'nın erime sıcaklığının (87,5oC) üzerine ısıtılması ve ardından 25oC'ye yavaş soğutulması sonucu elde edilmiştir. Fiziksel çapraz bağlı DNA jellerinin özelliklerini mukayese etmek amacıyla kimyasal çapraz bağlı DNA jelleri de bu tez kapsamında sentezlenmiştir. Kimyasal çapraz bağlı DNA hidrojelleri NaBr çözeltisi içerisinde çözülen DNA'nın, N,N,N',N'-tetrametiletilendiamin (TEMED) katalizörlüğünde ve etilenglikoldiglisidileter (EGDE) çapraz bağlayıcısı ile 50oC'de çapraz bağlanma reaksiyonları sonucu elde edilmiştir. Elde edilen fiziksel ve kimyasal çapraz bağlı DNA hidrojellerinin farklı tuz konsantrasyonlarında şişme ve salım davranışları incelenmiş, mekanik ölçümleri yapılmıştır. Kimyasal çapraz bağlı jellerin DNA salımlarının çok az olduğu, fiziksel jellerin ise kolaylıkla kontrollü salım sistemlerinde kullanılabileceği görülmüştür. Diğer yandan reolojik ölçümler ile ısıtma-soğutma çevrimlerine tabi tutularak kPa boyutlarında elastik modüle sahip fiziksel DNA hidrojelleri elde edilmiştir. Sentezlenen fiziksel jeller UV ve ATRFTIRölçümleri ile karakterize edilerek çifte sarmal yapıda olduğu ortaya konmuştur.

Abstract:
All living cells contain deoxyribonucleic acid (DNA) molecules carrying genetic information in their base sequences. DNA is a biopolymer composed of building blocks called nucleotides consisting of deoxyribose sugar, a phosphate group and four bases, adenine, thymine, guanine, and cytosine. DNA has a double-helical conformation in its native state which is stabilized by hydrogen bonds between the bases attached to the two strands. When a DNA solution is subjected to high temperatures, the hydrogen bonds holding the two strands together break and the double helix dissociates into two single strands having a random coil conformation. This transition from double-stranded (ds) to single-stranded (ss) DNA is known as denaturation or melting and can be reversed by slow cooling of dilute DNA solutions. DNA denaturation was extensively investigated in the past few decades as it gives useful information on DNA regarding its structure and stability. Typically, melting curves of DNA are obtained by monitoring the UV absorption at 260 nm. The disruption of base stacking decreases the electronic interaction between the bases so that it becomes easier for an electron to absorb a photon. Thus, denaturation leads to the hyperchromic effect, i.e., the increased absorption of light.At concentrations below the critical overlap concentration, DNA form viscous structures in aqueous solutions, while at high concentrations, DNA molecules overlap and entangle to form a weak gel. Only a few reports exist in the literature on the viscoelastic properties of DNA solutions. The linear viscoelastic moduli of DNA solutions have been measured by Mason et al. in the concentration range 0.1-1.0% w/v. It was shown that the solutions in saline buffer behave as an entanglement network of semiflexible coils exhibiting an elastic modulus and a crossover frequency that vary with concentration according to known scaling laws. Sun et al. showed that heating of semidilute solutions of DNA (0.5% w/v) leads to a decrease of the elastic modulus, indicating that the rigid-rod-like DNA molecules lost their rigidity due to the dissociation of the two strands. Thus, the semiflexible ds-DNA consisting of fragments of about 150 base pairs behaving as rigid segments becomes flexible on heating so that the viscosity of the solution decreases. The breakup of the DNA base pairs begins at a temperature as low as 50 °C, which is well below the chain melting temperature (87 °C).DNA hydrogel is a network of cross-linked DNA strands swollen in aqueous solutions. Such soft materials are a good candidate to make use of the characteristics of DNA such as coil-globule transition, biocompatibility, selective binding, and molecular recognition. DNA hydrogels were prepared starting from branched DNA molecules via ligase-mediated reactions. These hydrogels can also be prepared by cross-linking of DNA in aqueous solutions using diepoxides as chemical crosslinking agents. Epoxide groups can react with the amino groups on the nucleotide bases to form interstrand cross-links leading to the formation of a three dimensional xviiiDNA network. Our research group has recently focused on developing responsive DNA hydrogels with a wide range of tunable properties such as the conformation of the network strands, viscoelasticity, nonlinear elasticity, and porosity. It was shown that DNA can also be physically cross-linked by subjecting DNA solutions to heating-cooling cycles. Heating of semidilute solutions of ds-DNA above its melting temperature results in the dissociation of the double helix into flexible single strand fragments. On cooling back to the room temperature at a slow rate, the dissociated strands cannot re-organize to form the initial double stranded conformation. Hence, the hydrogen bonds formed between strands belonging to different ds-DNA molecules act as physical junction zones leading to the formation of gels with a modulus of elasticity between 101and 104Pa. The effects such as the concentration of DNA solution, pH, the presence of a cross-linker, and the duration of the heating period on the viscoelastic properties of the physical gels were investigated before. In the present study, the swelling behavior of both physically and chemically crosslinked DNA hydrogels was investigated in water and in aqueous salt solutions. The aim is to compare the swelling behavior of DNA hydrogels with that of synthetic polyelectrolyte hydrogels. DNA gels were made from deoxyribonucleic acid sodium salt from salmon testes. According to the manufacturer, the % G-C content of the DNA used is 41.2 %, and the melting temperature is reported to be 87.5oC in 0.15 M sodium chloride plus 0.015 M sodium citrate. Physical gels were prepared by subjecting aqueous solutions of 5 % DNA to heating–cooling cycles between below and above the melting temperature of DNA. Such physical gels were prepared before, on a small scale, between the parallel plates of rheometer, but their swelling behavior and stability in aqueous media have not been investigated. It was observed that, although the initial DNA solution flowed under gravity, the physical gels formed after the cycle preserved their shapes and they exhibited an elastic modulus of 2.1 kPa after a relaxation time of 3 sec. The conformation of DNA strands in the physical gels was investigated by ATR-FTIR measurements. It was found that the intensity of the characteristic ds-DNA peaks remain unchanged after gelation indicating that the DNA strands in the physical gels are in double stranded conformation. The conformation of DNA before and after heating-cooling cycle was also tested by determining the molar extinction coefficient of DNA, isolated from the gels, at 260 nm. Absorbance of DNA solutions at 260 nm is known to increase as the structure of DNA transforms from double to single strand; in case of complete denaturation, it increases 40 % due to the change of the molar absorption coefficient of the aromatic rings. The results show that the molar extinction coefficient of DNA is constant before and after gelation supporting that the physical gels consist of mainly ds-DNA strands. Physical DNA gel samples were subjected to swelling tests in aqueous NaBr solutions of various concentrations (Csalt) between 0 and 10-1 M. In salt solutions below 10-2 M NaBr, the relative weight swelling ratio mrel first increases with increasing swelling time, and after attaining a maximum value, it again decreases. By UV-vis measurements conducted on external salt solutions, it was found that the decrease in mrel is related with the disintegration of DNA chains from the gel network. While about 80 % of ds-DNA in the physical gels released within one day in 0 – 10-4 M NaBr, a much slower release over a time scale of many days was achieved at higher salt concentrations. Thus, the hydrogen bonds acting as physical cross-link zones between ds-DNA strands are destroyed during the expansion of the gels in dilute salt solutions. xixHowever, at higher salt concentrations, the gels immersed in solutions remain stable over many days. For example, in 10-2 M NaBr, the gels dissolved after about 16 days while in 10-1 M NaBr they remained stable over the whole swelling time studied (42 days). The results thus show that both the release rate and the released amount of DNA could be adjusted by the salt concentration in the solution. This also suggests that the physical DNA gels prepared without a cross-linker can be used for the controlled release of ds-DNA in aqueous solutions.Chemical DNA hydrogels were prepared by the solution cross-linking of DNA using ethylene glycol diglycidyl ether (EGDE) cross-linker. The gel samples were subjected to swelling tests in aqueous NaBr solutions of various concentrations Csaltbetween 0 and 1 M. The gel samples swell to equilibrium state within 1 to 2 days while at longer times, the gel mass remains constant. This is in contrast to the physical gels, and indicates the stability of the chemically cross-linked DNA network structure. The results also show that, below 10-4 M NaBr, the equilibrium swelling ratio of gels remains almost unchanged while further increase in Csalt leads to a rapid deswelling. The decreased swelling ratio of DNA hydrogels with increasing salt concentration is due to decrease in the concentration difference of counterions inside and outside the gel. The fixed phosphate residues of DNA strands confined to the gel phase require equal number of counterions (Na+) to stay within the gel to achieve electroneutrality.In conclusion, physical DNA gels generated via heating-cooling cycle of 5 % aqueous DNA solutions are not stable in water, indicating that the physical crosslinks are destroyed during the swelling process. It was found that both the release rate and the released amount of DNA from the physical gels can be adjusted by changing the salt concentration in the external solutions. In this way, the release of ds-DNA molecules from the gels can be varied between a few hours and tens of days. This suggests that the physical gels can be used for the controlled release of DNA in aqueous media. In contrast, chemically cross-linked DNA gels formed using EGDE cross-linker are stable in water and thus, their swelling ratios could be adjusted by the amount of DNA at the gel preparations.