Sürfaktan Varlığında Sentezlenen Poli (n-[3-(dimetilamino)propil] Metakrilamid) Hidrojellerinin Mekanik Dayanımları

Abstract

Hidrojeller polimer zincirlerinin fiziksel ya da kimyasal olarak birbiri ile bağlanmasıyla oluşan ve şişmiş halde suda çözünmeden kalabilen polimerik ağ yapılardır. Bu nedenle özellikle biyoteknolojik uygulamalarda ilaç salımı, canlı hücre sistemlerinin simülasyonu ya da membran sistemlerinde ilgi çekici malzemelerdir. Hidrojel sistemlerinin canlı fizyolojisinde kullanılması için ortamın pH’ ına, vücuttaki kan basıncına dayanıklı olması ve besin- ilaç taşınması gibi görevleri yerine getirebilmesi için şişme derecesinin yüksek olması gerekmektedir. Hidrojeller, su tutma kapasitesine sahip hidrofilik karakterli malzemeler olduklarından şişme dereceleri yüksek ancak mekanik dayanımları düşüktür. Bir hidrojelin mekanik dayanımı hidrojeli oluşturan polimerler arasındaki çapraz bağın arttırılmasıyla, sentezi sırasında ortama hidrofobik bir komonomer ilavesiyle arttırılabilir ancak yapılan bu modifikasyonlar hidrojelin şişme derecesini azalmaktadır. Literatür bulguları incelendiğinde sentez sırasnda ortama inert molekül ilavesi ile hidrojelin morfolojisi değiştirilerek hem yapının mekanik dayanımınının hem de şişme derecesinin arttırıldığını rapor eden çalışmalar bulunmaktadır. Polimer sistemlerinin biyoteknolojik uygulamalarda kullanımının yaygınlaşmasıyla birlikte hidrojel dizaynında hidrofobik karakterler canlı fizyolojisinde toksisteye neden olduğundan istenmememektedir. Bu nedenle yapılan araştırmalar hidrofobik etkinin az olduğu hidrojel sistemlerinin geliştirilmesine doğru kaymaktadır. Bu tezde de, hidrolize karşı dirençli olması, vücudumuzda doğal olarak bulunan antimikrobiyal peptidlerdeki (AMP) amino asitlere yapısal benzerliği, fizyolojik pH’ta pozitif yüklü halde bulunması (PDMAPMAAm için pKa ~ 8.8), yapısındaki katyonik ve hidrofobik gruplar nedeni ile antimikrobiyal etki göstermesinin beklenmesi nedeni ile, PDMAPMAAm hidrojelleri sentezi yapılmıştır. Biyouyumlu bir malzeme olduğu bilinen bu hidrojellerin en zayıf özelliği mekanik dayanımlarıdır. Bu çalışmada, (i) ağırlıkça %20- %40 konsantrasyonları arasında değişen katyonik sürfaktan N-setil-N,N,N-trimetilamonyum bromür (CTAB) kullanılarak serbest radikal çözelti polimerizasyonu ile sentezlenecek olan PDMAPMAAm hidrojellerinin mekanik dayanımlarındaki değişimi/iyileşmeyi mekanik test cihazındaki sıkıştırma modül ölçümleri ile incelerken, liyotropik sıvı kristal davranışı sergileyen CTAB’ın yapıya mikro boyutdaki etkileri polarize optik mikroskop (POM) ve X-ışınları kırınımı (XRD) ile araştrılmış, gözlenen hekzagonal ve lamaler arafazların hidrojelin mekanik dayanımına, şişme derecesine ve antibakteriyel etkinlik üzerine etkileri incelenmiştir. Ayrıca, sentezlenen hidrojellerin gravimetrik/volumetrik şişme dereceleri, CTAB konsantrasyonu-iletkenlik ilişkisinin incelenmesi sonucunda iletkenliğin konsantrasyona ve sıcaklığa bağlı olduğu gözlenmiş ve CTAB’ ın Kraft sıcaklığı belirlenerek bu sıcaklık sonrası oluşan misellerin düzenlenerek oluşan solucanımsı (worm like) ve çubuğumsu (rod like) miselleri, bunların yığışımı ile oluşan lamelar ve hekzagonal arafazların hidrojelin mekanik dayanımı üzerinde etkili olduğu ve şişme derecesinin kontrol edilebildiği gözlenmiştir. Ayrıca, izotropik PDMAPMAAm hidrojeli ile birlikte mekanik dayanımları ve şişme davranışları iyileştirilmiş hem kuvaternize amonyum grubu ve hem de 16 ve 18 karbonlu alkil zincirleri taşıyan, CTAB varlığında sentezlenmiş anizotropik PDMAPMAAm hidrojellerinin disk difüzyon yöntemi ile E. Coli üzerindeki antimikrobiyal etkileri incelenmiş, CTAB varlığında sentezlenen PDMAPMAAm hidrojellerinin mekanik dayanımları iyileşirken, antibakteriyel etkinliklerinin yapısal düzenlemelerin etkisi ile izotropik hidrojellere kıyasla azaldığı gözlenmiştir.

Hydrogels are crosslinked polymeric network structures having large amount of water absorption capacities. They are useful in various biotechnological applications including contact lenses, tissue scaffolds, separation membranes, and drug delivery systems due to their structural and functional features such as high water content, biocompatibility, and porosity. High swelling degrees and mechanical strength are necessary to use them in physiological body fluids. However, high swelling degrees can cause mechanical weakness. To overcome the weakness in mechanical properties of hydrogels, some structural modifications can be made like comonomer addition to main polymer chain, using a different crosslinker, presence of secondary polymer network and addition of nanoparticles during the synthesis of hydrogel. Amphiphilic molecules called surfactants have a great potential as material because of their liquid crystal behaviours. Amphiphilic molecules contain two parts in their natures. One of them is polar head group which has ionic character in solution, and another is non polar tail group that bear hyrophobic hydrocarbon chain. Amphiphilic molecules are classified as nonionic, cationic, anioic. For example, N-cetyl-N,N,N, trimethylamonium bromide (CTAB) is a cationic surfactant, sodium dodecyl sulfat (SDS) is an anionic surfantant and n-dodecyl tetra (ethylene oxide) (C12E4) is a non-ionic surfactant. When their concentrations are extremely low, amphiphiles form to monolayer between air and water. With increasing concentration, these ordered structures change to agregates in water. In the mean time hydrophobic parts of surfactant move away from water molecules and hydrophilic part of surfactans interact with water molecules. This phenomenon is called “micelle formation”. Self-assemblies of surfactant depend on their critical micelle concentrations (CMC). Below CMC, surfactant molecules dispersed randomly as monomers, whereas above the CMC, the molecules become to form micelles. Another important parameter in the micelle formation is packaging paremeter. According to packaging parameter micelles form to spherical, cylindrical, planer or inverted micelles. Above CMC amphiphiles show isotropic dispersion of spherical micelles. As surfactant concentration is increased, micelles form to cylindircal micelles, which create a hexagonal order and/or lamellar order. Surfactants can be used as a template during hydrogel synthesis. These clusters in the surfactants are defined as lyotropic liquid mesophases. Mechanical strength and swelling behaviour of a hydrogel can be controlled by using surfactants as a template during its synthesis. As to the results presented in literature, these ordered structures can control hydrogel porosity. Antimicrobial peptides (AMPs) show great potential as alternative antibacterial agents to conventional antibiotics as they can selectively bind and eliminate pathogenic bacteria without any harming eukaryotic cells. However, they are expensive materials to produce, therefore it is necessary to develop synthetic macromolecules to mimic AMPs behavior. According to the literature results, primer amin group is the more effective than secondary and tertinary group on bacteria. N-[3-(dimethylamino)propyl] methacrylamide (DMAPMAAm) is similar to structural units in antimicrobial peptides due to the chemical structure having hydrophobic and hydrophilic moieties and pKa (~8.8) value in the physiological pH range (pH 6-8). However, its mechanical strength in swollen state is not sufficient for use in drug-delivery formulations and antimicrobial applications. This work reports the syntheses of PDMAPMAAm hydrogels in the presence of cationic surfactant, N-cetyl-N,N,N, trimethylamonium bromide (CTAB) by using two different mixing procedure: mechanical (homogenizer) and magnetic stirring. They were prepared by free-radical solution polymerization in distilled-deionized water (DDW) and, by using potassium persulfate (KPS) and N,N’-methylenebisacrylamide (BIS) as initiator and crosslinker, respectively, in prensence of various CTAB concentration from %20 to % 40. Textures of lyotrophic liquid crystalline (LLC) mesophases were investigated by using polarized optical microscopy (POM) and X-ray diffraction analysis (XRD). All the results were compared with the ones of isotropic PDMAPMAAm hydrogels. Compressive moduli, swelling behaviour and antibacterial properties of drug loaded and blank PDMAPMAAm hydrogels were measured by unaxial compression, gravimetry and inhibition zone tests, respectively. It was obseved that, the compression moduli of the swollen hydrogels after removal of surfactant molecules (DDW-swollen) were highly increase whereas isotropic hydrogels synthesized without CTAB exhibited fragile structure and higher water uptake. The compressive moduli of CTAB loaded-PDMAPMAAm hydrogels increased from ~ 3.0 kPa to 45.0 kPa even after removal of surfactant molecules (DDW-swollen) and, they withstood under a load of 5.0 N without breaking. Templated-PDMAPMAAm hydrogels were synthesized in the presence of three different concentration of cationic surfactant, CTAB (20, 25 and 30 wt %) by using three different concentration of DMAPMAAm (2.0, 3.0 and 4.0 mol/L) with two different mixing process. Their polarized optical microscope (POM) images before removal of LLC templates, i.e., CTAB molecules (in our case) exhibited the formation of hexagonal and lamellar mesophases. The formation of lyotrophic liquid crystal mesophases, resulted from the ordered/disordered arrengements of non-reactive CTAB molecules in the structures of PDMAPMAAm hydrogels, were depent on the content of N-cetyl-N,N,N, trimethylamonium bromide (in the range of 20.0 to 40.0 wt %), mixing type of pregel solution and reaction temperature. POM images of their aqueous CTAB solutions and CTAB-templated PDMAPMAAm hyrogels showed the formation of hexagonal and lamellar mesophases at 115 0C but at the room temperature, textures of both CTAB solutions and their corresponding hydrogels solidified in crystal form. Further, with increasing temperature, their solutions become clear. Their viscosities also increases due to formation of various aggregation forms. The conductivity-temperature curves and Krafft temperature/Krafft points on these plots supported the presence of temperature- and concentration- dependent micelle and vesicle formations. Antibacterial activities on E. Coli of PDMAPMAAm hydrogels were investigated by using disk diffusion method. At first, E. Coli was planted into petri dishes and then PDMAPMAAm hydrogels synthesized in the absence and the presence of CTAB molecules were placed into the drug solution for swelling equilibria for 48 hours. Diclofenac sodium (DFNa), gentamicin sulphate (GS) and ciprofloxacin (CP) were used as drug molecules for release experiment in petri dishes. After the placement of both isotropic/CTAB-loaded PDMAPMAAm hydrogels and their corresponding DFNA-, GS, and CP- loaded samples on the E.Coli – planted dishes, their inhibition zones were measured. The results indicated that their antibacterial effectiveness were dependent on the microstructures of the hydrogels synthesized in the absence and presence of CTAB as template rather than the type of loaded drugs.