Sıcaklık ve pH'ya duyarlı poliakrilik asit/pluronik içiçe geçmiş ağ yapıların sentezi ve karakterizasyonu
Synthesis and characterization of ph and temperature sensitive polyacrylic acid/pluronics F127 based interpenetrating hydrogels
Yazar: TUBA BAŞKAN
Danışman: PROF. DR. GÜLAÇTI TOPÇU ; PROF. DR. OĞUZ OKAY
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Bölümü / Kimya Ana Bilim Dalı
Derece: Yüksek Lisans, 2013
Konu: Kimya = Chemistry

Özet:
Hidrojeller, suda çözünmeden şişebilen katı ile sıvı arası davranış gösteren,  hidrofilik üç boyutlu polimerik ağyapıların suda şişmeleri ile oluşan malzemelerdir. Hidrojellerin şişme-büzülme, uyarılara cevap verme, sıvı-jel geçişleri gibi özellikleriyle birlikte proteinleri ve ilaçları dış çevreden korumaları biyomedikal ve biyoteknolojik alanlarda kullanılmasını sağlamıştır. Özellikle sıcaklığa ve pH'ya duyarlı hidrojeller, ayarlanabilir şişme ve ilaç salıverme özelliklerinden dolayı ilaç taşıyıcı sistemi olarak kullanmıştır. pH?ya duyarlı poliakrilik asit (PAAc) hidrojelleri büyük ölçüde lokal ve oral ilaç salım sistemlerinde kullanılan mukayapışkan özelliktedir. Sıcaklığa duyarlı polietilenoksit(PEO)-polipropilenoksit(PPO)-polietilenoksit(PEO) blok kopolimerleri yüzey aktif maddelerin önemli bir sınıfı olup deterjan, dispersiyon stabilizasyonu, köpükleşme, emülsifikasyon, yağlama gibi oldukça geniş endüstriyel alanlarda kullanılmaktadır. Ticari adı Pluronik veya Poloksamer olan amfifilik karakterdeki bu kopolimerlerin farmasötik alanda ilaç çözünümü ve ilacın kontrollü salımı gibi uygulamaları bulunmaktadır. PEO-PPO-PEO blok kopolimer çözeltilerinin en önemli iki özelliği sıcaklığa bağlı miselleşmesi ve jel oluşumudur.Gerek Pluronik ve gerekse Pluronik-PAAc kopolimerlerinin sulu çözeltileri, sıcaklığa ve pH'ya duyarlı olup bu özellikleriyle biyomedikal uygulamalarda kullanılmaktadır. Ancak bu tip polimer sistemlerinin uygulama alanında en büyük dezavantajı, mekanik dayanımlarının yetersiz oluşudur. 
İçiçe geçmiş ağyapılar (IPN), farklı birleşimliiki ya da daha fazla ağyapının moleküler boyutta birbirinin içine girmesiyle oluşmaktadır ve ancak ağyapılardan en az birisinin kırılmasıyla ayrılabilmektedir. IPN yapıları, birbirlerine uymayan özellikler veya fonksiyonlar taşıyan  iki ya da daha fazla grupların karışmasından ve bununla birlikte sinerjist özellik göstermesinden dolayı teknolojik olarak çok büyük ilgi görmektedir.Bu çalışma kapsamında, Pluronik jellerinin mekanik özelliklerini iyileştirmek amacıyla poliakrilik asit (PAAc) ile IPN hidrojelleri sentezlenmiştir.  Bu amaçla, akrilik asit (AAc) monomerinin bir çapraz bağlayıcı varlığında kopolimerizasyonu Pluronik F127'nin sulu çözeltilerinde gerçekleştirilmiş,karşılaştırmak amaçlı PAAc hidrojelleride sentezlenmiştir.Sentezlenen IPN ve PAAc hidrojellerin; pH'ya duyarlılığı şişme testleriyle, sıcaklığa duyarlılığı reometre cihazında yapılan ısıtma soğutma çevrim testleriyle belirlenmiştir. Mekanik özellikler üniversal test cihazında tek eksenli basma ve çekme testleriyle birlikte döngüsel basma testleri yapılarak incelenmiştir. 
Farklı pH'larda yapılan şişme testleri sonucunda, düşük pH değerlerinde, çapraz bağ yoğunluğu daha az olan IPN hidrojellerin PAAc hidrojellerine gore daha az şiştiği görülmüştür. Bu beklenmedik davranış, F127 moleküllerinin AAc'in dissosiasyon sabitini düşürmesiyle açıklanmıştır.Yüksek pH değerlerinde ise, AAc birimlerin iyonizasyon derecesinin artması ve hidrofobik assosiasyonların zayıflamasıyla düşük çapraz bağ yoğunluğuna sahip IPN hidrojelleri PAAc hidrojellerine gore daha fazla şiştiği gözlemlenmiştir.
Reometrik ölçümler sonucunda, F127varlığının PAAc hidrojelinin elastik modülünü düşürdüğü; buna karşılık viskoz modülünü ise arttırdığı gözlemlenmiştir.Viskoz modül değerlerindeki artış, F127 moleküllerin çapraz bağlı PAAc hidrojelinde moleküler seviyede enerji dağıtıcı mekanizma yaratmasıyla açıklanmıştır.F127 çözeltisinin ve hidrojellerin sıcaklığa bağlı davranışları incelendiğinde, sıcaklık arttıkça elastik modülün arttığı ve IPN hidrojellerinde AAc konsantrasyonu arttıkça elastik modül değerlerindeki değişimin azaldığı görülmüştür.Sıcaklık ile elastik modül değerleri arasındaki ilişki, sıcaklığın artmasıyla misel sayılarındaki artış ve artan misellerarası entanglementların(dolaşıklık)fiziksel çapraz bağ gibi davranmasıyla açıklanmıştır.
Üniversal test cihazında yapılan tek eksenli basma ve çekme testleri sonucunda, tüm AAc yüzdelerinde IPN hidrojellerin, PAAc hidrojellerine göre daha yüksek gerinim oranına sahip oldukları görülmüştür. Bu davranış, IPN hidrojellerinde bulunan F127 moleküllerinin oluşturduğu asosiasyonlarla yapının tokluğunun artıp mekanik özelliklerin iyileştiğini göstermiştir.Tek eksenli döngüsel basma testleri sonucunda, yüksek gerinimlerde, IPN hidrojellerin histeresis enerjisinin PAAc hidrojellerine göre daha yüksek olduğu görülmüştür.Bu durum, IPN hidrojellerinde daha fazla sayıda fiziksel bağların olmasıyla birlikte hem iyonik kümelerin hem de hidrofobik asosiasyonların varlığıyla açıklanmıştır.
Bu tez çalışması sonucunda, sentezlenen IPN hidrojellerin sıcaklığa ve pH'ya duyarlı olduğu görülmüştür.Bununla birlikte;içiçe geçmiş ağyapı senteziyle, mekanik özellikleri zayıf olan F127 hidrojellerinin mekanik özellikleri iyileştirilmiştir.Böylece, F127 hidrojellerin fizyolojik ortamda kolayca dağılabilmesinden dolayı kısa süreli tedavilerde kullanılma kısıtlaması ortadan kaldırılmıştır.
Bu çalışma kapsamında sentezlenen Pluronik F127 / PAAc IPN hidrojelleri son yıllarda yayınlanan iki ayrı çalışmada rapor edilmiş olup sadece kontrollu salım davranışları belirtilmiştir. IPN oluşumu ve oluşan IPN hidrojellerin reolojik ve mekanik özellikleri ile pH ve sıcaklığa duyarlılığı ilk olarak bu tez çalışmaları kapsamında ortaya konulmuştur.

Abstract:
Hydrophobic interactions play a dominant role in the formation of large biological systems. These interactions can be generated in synthetic polymer systems by incorporation of hydrophobic sequences within the hydrophilic polymer chains.  Aqueous solutions of hydrophobically modified hydrophilic polymers constitute a class of soft materials with remarkable rheological properties. Above a certain polymer concentration, the hydrophobic groups in such associative polymers are involved in intermolecular associations that act as reversible breakable crosslinks creating a transient 3D polymer network.  Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers, known under the trade name Pluronics, are typical amphiphilic polymers with associative properties. These copolymers may undergo thermoreversible micellization and gelation in aqueous solutions via associations of hydrophobic poly(propylene oxide) (PPO) blocks. PPO center blocks form the core of Pluronic micelles while the relatively hydrophilic poly(ethylene oxide)(PEO) blocks forming the micelle shells interact with those of neighboring micelles. As the temperature is increased, the number of Pluronic micelles also increases leading to the formation of thermoreversible hydrogels via intermicellar entanglements between PEO segments.  
In the Pluronic family, the most extensively studied member is F127 (PEO99-PPO65-PEO99) due to its good solubility in water and a high capacity for hydrophobic association through the relatively long PPO block. Above a certain concentration and temperature, aqueous solutions of F127 exist as spherical micelles with an aggregation number of about 50. The degree of overlap of the micelle shells depends on F127 concentration; at or above 18 % F127, the spherical micelles pack onto a simple cubic lattice to form physical gels. Pluronic F127 has attracted much attention as injectable drug delivery systems. The sol state of F127 in aqueous solutions at room temperature facilitates incorporation of bioactive molecules, while the gel state at physiological temperature allows F127 hydrogels to serve as a drug-delivery depot. One limitation of F127 hydrogels is that they are mechanically weak and easily dissolve in physiological environments, which limit their use in load-bearing applications. Several efforts have been made recently to improve the mechanical performance of F127 hydrogels. For example, acrylate-functionalized F127 has been polymerized to obtain chemically crosslinked F127 hydrogels. It was also shown that the crosslinking of F127-diacrylates in the presence of clay nanoparticles produces high-toughness nanocomposite hydrogels.In ethoxysilane-capped Pluronic copolymers, the ethoxysilane groups hydrolyze over time to form silanol groups which covalently crosslink the copolymers. Alternatively, amine-terminated Pluronics can be grafted with hyaluronic acid to form hydrogels with a reduced rate of dissolution due to the hyaluronic acid grafts.  
In order to prevent dissolution of F127 hydrogels in aqueous media and to improve their mechanical properties, we describe here the preparation of interpenetrating polymer network (IPN) hydrogels composed of F127 and polyacrylic acid (PAAc) network chains. The design principle of the present IPN hydrogels bases on the fact that polyethers form long-lived macroradicals in the presence of radical initiators.  It was reported that the free - radical polymerization of monomers such as acrylic acid (AAc) with the chain transfer to F127 results in grafting of PAAc chains onto the Pluronic backbone. F127-g-PAAc copolymers have unique graft-comb like structure whereby PAAc chains were attached to PPO segments of F127 via C-C bonding.  The graft copolymers, as linear chains or, as intramolecularly crosslinked molecules (microgels), are capable of self-assembly in response to temperature changes in aqueous media.
To obtain high-toughness IPN hydrogels with pH- and temperature-sensitive properties, we performed the free - radical crosslinking copolymerization of the AAc monomer and N,N'-methylenebis(acrylamide) (BAAm) crosslinker in aqueous 20 w/v % F127 solutions. Gelation reactions were monitored by classical rheometry using oscillatory deformation tests. For comparison, hydrogels were also prepared in the absence of F127. In the following, hydrogels formed with and without F127 will be called IPN and PAAc hydrogels, respectively.
It was found that the presence of F127 within the gel network slightly decreases the elastic modulus while the loss factor significantly increases, revealing increasing energy dissipation in IPN hydrogels. Thus, viscoelastic properties of the chemical PAAc hydrogel significantly change after incorporation of F127 into the gel network.  Increasing AAc % in the feed from 10 to 30 w/v % also increased the elastic modulus of the hydrogels while their loss factor decreased; indicating that less energy is dissipated at high AAc contents. Moreover, no gel was obtained when AAc % was decreased to 5 % probably due to the extensive chain transfer reactions in the presence of F127. The incorporation of F127 into the gel network via covalent bonds was checked by the measurement of the gel fraction Wg, the mass of crosslinked, water-insoluble polymer obtained from one gram of AAc - F127 mixture in the feed.  Without F127, gel fraction Wg was equal to unity over the whole range of AAc concentration.  In the presence of F127,  Wg was also found to be unity at 20 and 30 w/v % AAc in the feed, indicating that all F127 molecules were incorporated into the polymer matrix via chain transfer reactions. At 10 w/v % AAc, the gel fraction Wg decreased to 0.6; assuming that the conversion of AAc to the crosslinked polymer is complete, this means that about 40 % of F127 in the gelation solution become part of the hydrogel network at this AAc concentration.  
Elasticity measurements show that the shear modulus of IPN hydrogels is smaller than PAAc hydrogels over the whole range of AAc content. Assuming affine network behavior, the effective crosslink densities ?ewere calculated from the moduli data of both IPN and PAAc hydrogels. IPN hydrogel exhibited a lower effective crosslink density as compared to the corresponding PAAc hydrogel formed in the absence of F127 molecules. This reduction in ?e may be attributed to the steric effect of F127 on the gelation kinetics of vinyl-divinyl monomer copolymerization. Thus, F127 molecules attached to the growing PAAc chain radicals during gelation may reduce the reactivity of the pendant vinyl groups to form effective crosslinks.  Moreover, formation of shorter primary chains due to the chain transfer to F127 may also be responsible for this behavior. Since the lower the crosslink density, the higher the swelling ratio, one may expect a higher degree of swelling of IPN hydrogels as compared to PAAc hydrogels. However, an opposite behavior was observed in the swelling behavior of IPN hydrogels. Although IPN hydrogels are less crosslinked, they exhibited a lesser degree of swelling at pH < 9 as compared to the corresponding PAAc hydrogels. We attribute this behavior to the decrease of the dissociation degree of AAc units in the network chains due to the presence of F127 molecules. It was indeed observed that pKa of F127-g-PAAc microgels is larger than pKa of PAAc. This difference in pKa was explained by the formation of hydrophobic associations between PPO segments of the microgels in aqueous solutions.  Such associations alter electrostatic charge density and the availability of the carboxyls on the PAAc chains to neutralization, which may lead to a decrease in the swelling ratios of the hydrogels containing F127. However, at high pH, increasing degree of ionization of AAc units also increases the osmotic pressure exerted by the mobile counterions leading to the weakening of the hydrophobic associations and to gel swelling. This was supported by the fact that at pH > 9, IPN hydrogels swell more than PAAc gels due to their lower crosslink densities.   
Mechanical properties of the hydrogels were determined by elongation and compression tests.IPN hydrogels exhibited a higher strain at break and a higher degree of toughness when compared to the PAAc gel controls.For example, PAAc hydrogel formed at 10 w/v % AAc fractures under a compression of 0.2 MPa at 78 % strain while the corresponding IPN hydrogel sustains up to 7 MPa compressions at 98 % strain, leading to an increase of toughness from 31 to 335 kJ/m3.. The improvement in the mechanical performance of IPN hydrogels is due to the increased associativity in the gel network so that more energy is dissipated under deformation at large strains. Moreover, for PAAc hydrogels, the compression ratio at break and the compressive strength are independent on the strain rate in the range investigated, while in IPN hydrogels, they both increase as the strain rate is increased. These results highlight the dynamic properties of IPN hydrogels and in accord with the rheological test results.  
The large strain properties of IPN and PAAc hydrogels were compared by cyclic compression tests conducted up to a strain below the failure. In both IPN and PAAc hydrogels, the loading curve of the compressive cycle was different from the unloading curve indicating damage in the gels and dissipation of energy during the cycle.  The reversibility of loading/unloading cycles was observed in all gel samples with or without F127. This suggests the existence of reversible breakable bonds in both PAAc and IPN hydrogels. The energy Uhys dissipated during the compression cycle was calculated from the area between the loading and unloading curves. It was foundthat the hysteresis energy Uhys at large strains is much larger in IPN hydrogels as compared to PAAc hydrogels.  Because Uhyscan be interpreted as the dissociation energy of physical bonds broken down during the compression cycle, this reveals existence of a larger number of such physical bonds in IPN hydrogels. In PAAc hydrogels with negligible viscous properties, the appearance of hysteresis is due to the reversible formation of ionic clusters under large strain, as identified recently. In IPN hydrogels, Uhys is much larger due to the presence of both ionic clusters and hydrophobic associations of F127 molecules. Thus, the larger number of breakable bonds in IPN hydrogels is responsible for their enhanced mechanical properties.
The thermal behavior of IPN hydrogels was investigated by subjecting the gel samples to heating - cooling cycles between 25 and 50oC, during which the dynamic moduli of the gels were monitored as a function of temperature. For comparison, we first investigated the thermal behavior of 20 w/v % F127 solution during this cycle.   Before heating, the solution at 25oC shows a liquidlike response, i.e. G'' exceeds G' over the whole range of frequencies investigated, indicating that F127 chains in the solution mainly exist as unimers. During heating of the solution and particularly between 28 and 33oC, both moduli rapidly increase and, gelation occurs at 28oC as evidenced by the decrease of the los factor tan?   below unity. Gelation of F127 solution is due to the increased hydrophobicity of PPO blocks with rising temperature, leading to the destruction of the cagelike water structure surrounding F127 molecules. This dehydration of F127 leads to the formation of spherical micelles with PPO blocks forming the core surrounded by a shell of PEO blocks.  As the temperature is increased, the number of the micelles also increases making the intermicellar distances shorter so that the intermicellar entanglements acting as physical crosslinks lead to F127 gelation. More than 4 orders of magnitude increase of the elastic modulus during heating suggest the existence of a significant number of such entanglements serving as crosslinks at the time scale of the experiments (about 0.2 s). Moreover, the fact that the viscous modulus also increases significantly in the same range of temperature reveals that the intermicellar frictions of close packed F127 micelles significantly contribute to their viscous behavior.  
The results also show that the sol-gel transition phenomenon of 20 % F127 solution is not reversible, i.e., at the end of the heating-cooling cycle, the system does not return to its initial state and remains as an elastic F127 mesh. This behavior is mainly attributed to the slow relaxation of intermicellar entanglements due to the high viscosity of the solution. Since F127 concentration used in the present study (20 %) is much larger than the cmc value of F127 at 25oC (~0.7 %), one may expect that the gel of close packed F127 micelles formed at a high temperature cannot reorganize to form the initial conformation upon cooling back to 25oC.  
IPN and PAAc hydrogels were also subjected to the same heating-cooling cycle.  The viscous modulus G'' and tan   of PAAc hydrogel remain almost unchanged over the whole temperature range while its elastic modulus G? slightly increases due to entropic effects. In contrast, the dynamic moduli of IPN hydrogel significantly increase with rising temperature indicating that the addition of F127 generates temperature sensitivity in PAAc gels. All IPN hydrogels exhibited temperature sensitivity with an increase in the dynamic moduli on rising the temperature, revealing formation of hydrophobic associations within the gel network. The temperature sensitivity of IPN hydrogels was also observed after their equilibrium swelling in water. As compared to F127 solution, the extent of the moduli variations is however limited due to the crosslinked network structure and, due to the structural constraints to F127 segments by the attached PAAc network chains.