Deneysel ve modelleme yöntemleri ile poliüretan filmlerdeki pürüzlülük ve kristalinitenin fibrinojen adsorpsiyonu üzerindeki etkilerinin belirlenmesi
Determining the effects of roughness and crystallinity on fibrinogen adsorption on polyurethane films with experimental and modeling methods
Yazar: GİZEM KELEŞ
Danışman: DR. ÖĞR. ÜYESİ AYŞE ÖZGE KÜRKÇÜOĞLU LEVİTAS
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Mühendisliği Ana Bilim Dalı / Kimya Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2019
Konu: Kimya Mühendisliği = Chemical Engineering

Özet:
Polimer malzemelerin kullanıldığı önemli uygulama alanlarından biri de biyomedikal alanıdır. Biyomalzemeler kanla temas ettiklerinde ilk gerçekleşen olay kan proteinlerinin katı (polimer) - sıvı (kan) arayüzeyinde adsorpsiyonu olduğundan, biyomedikal uygulamalarda tercih edilen biyomalzemelerin daha kullanışlı hale getirilebilmesi için polimer yüzeye protein adsorpsiyon mekanizmasının anlaşılması ve protein adsorpsiyonunun kontrol edilmesi oldukça önemli bir konudur. Protein adsorpsiyonunu birden fazla faktör etkilemektedir. Proteinin, çözeltinin ve adsorbent yüzeyinin özellikleri protein adsorpsiyonu üzerinde sinerjik bir etkiye sahip olduğundan protein adsorpsiyon olayını kontrol edebilmek zorlaşmaktadır. Proteinlerin büyüklüğü, esnekliği ve hidrofilikliği adsorpsiyonu etkileyen protein özellikleridir. Ayrıca, pH, sıcaklık, iyonik kuvvet, kimyasal bileşim ve konsantrasyon gibi çözelti özellikleri ile polimer yüzeyinin kimyasal, geometrik ve elektriksel özellikleri protein adsorpsiyonunu etkileyen etmenlerdendir. Bu çalışmada, biyouyumlu bir polimer olan poliüretan (PU) tercih edilmiş olup pürüzlülük ve kristalinitenin fibrinojen adsorpsiyonu üzerindeki etkileri hem deneysel hem de moleküler modelleme ve simülasyon yöntemleri ile belirlenmiştir.
Deneysel çalışma kapsamında, farklı yüzey özelliklerine sahip PU film sentezleri yapılarak karakterizasyon çalışmalarının tamamlanmasının ardından, fibrinojen proteininin PU yüzeylere adsorpsiyonu incelenmiştir. PU'nun mekanik özelliklerinin ve kan uyumluluğunun yüksek olması ile fiziksel ve kimyasal özelliklerinin esnek olmasının verdiği avantajların etkisiyle, biyomedikal alanındaki uygulamalarda biyomalzeme olarak kullanımı sıklıkla tercih edilmektedir. PU'nun özellikleri temel olarak reaktif grupların içeriğine, yapıdaki çapraz bağlanma derecesine ve reaksiyona giren monomerlerin birbirlerine dolanma seviyelerine bağlıdır. PU sentezinde petrol bazlı polioller kullanılabileceği gibi bitkisel yağlar da kullanılabilmektedir. Son zamanlarda, çevresel kaygı ve ekonomik sebeplerden dolayı sentezde bitkisel yağlar gibi yenilenebilir ham maddelerin kullanımına önem verilmektedir. Ayrıca, bitkisel yağların düşük toksisitede, yüksek saflıkta ve ucuz olmaları da tercih edilmelerini arttırmaktadır. Bitkisel bir poliol olan hint yağı, PU sentezinde çapraz bağlayıcı olarak kullanılırken, 1,4-bütandiol (BDO) zincir uzatıcı, polietilen glikol (PEG) poliol kaynağı ve hekzametilen diizosiyanat (HDI) izosiyanat kaynağı olarak kullanılmıştır. Temel kan proteinleri albümin, hemoglobin, gamma globulin, fibrinojen ve ferritindir. Yapılan önceki bir çalışmada, fibrinojenden daha küçük ve küresel bir yapıya sahip olan albümin proteininin çeşitli PU yüzeylere adsorpsiyon olayı incelenmiştir. Fibrinojenin anizotropik ve esnek yapısının adsorpsiyona etkisini ortaya çıkarmak için bu çalışmada fibrinojen proteininin çeşitli PU yüzeylere adsorpsiyon analizleri yapılmıştır. Sentezlenen PU filmlerin karakterizasyonları, FTIR, DSC, su ile temas açısı, şişme ve AFM analizleriyle yapılmıştır.
Deneysel çalışmalar sonucunda, polimerin hidrofilikliğinin, kristalinitesinin ve yüzey pürüzlülüğünün adsorpsiyonu belirleyen önemli etmenler olduğu anlaşılmıştır. Fibrinojen anizotropik ve esnek bir yapıya sahip olduğundan simülasyon çalışmasına fibrinojenin esnek bölgelerinin ve menteşe hareketlerinin belirlenmesiyle başlanmıştır. Fibrinojenin esnek kısımlarını ve menteşe hareketlerini belirlerken Gaussian ağ modelinden yararlanılmıştır. Esnek kısımlar fibrinojenin orta ve iki ucunda bulunduğu için yapı modellemesi yapılırken fibrinojen, birbirlerine yaylarla bağlı üç nodül şeklinde modellenmiştir. Fibrinojen modelinin belirlenmesinin ardından, yüzey pürüzlülüğü ve kristalinitenin adsorpsiyon üzerindeki etkileri Brownian dinamiği simülasyonu ile incelenmiştir. Yüzey kristalinitesinin protein adsorpsiyonuna etkisini araştırabilmek için adsorpsiyon bölgelerinin sıralı ve gelişigüzel yer aldığı iki çeşit düz yüzey modellenmiştir. Ayrıca, farklı pürüzlülük ve pürüz derinliklerine sahip adsorpsiyon bölgelerinin gelişigüzel dağılım gösterdiği üç tip pürüzlü yüzey modellenerek yüzey pürüzlülüğünün fibrinojen adsorpsiyonuna etkisi Brownian simülasyon yöntemi yardımıyla analiz edilmiştir. Simülasyon çalışmaları sonucunda, kristalinitenin, yüzey pürüzlülüğünün ve adsorplanan fibrinojenin oluşturduğu sterik engelin adsorpsiyonu belirlediği anlaşılmıştır. Deneysel çalışmadan elde edilen sonuçlarla simülasyon sonuçları karşılaştırıldığında, yüzey pürüzlülüğünün artmasıyla fibrinojen adsorpsiyonunun artış gösterdiği ortak sonucuna varılmıştır.

Abstract:
Since polymeric materials are synthesized from small molecules called monomers by creating chemical bonds, these materials have long chain structures. Polymers constitute a large part of the materials, and synthetic polymers as biomaterials are generally preferred in biomedical applications. There is a large variety of synthetic polymers, and some of them can be used as biomaterials including polyethylene (PE), polyurethane (PU), polytetrafluoroethylene (PTFE), polyacetal (PA), polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), silicone rubber (SR), polysulfone (PS), polyetheretherketone (PEEK), poly (lactic acid) (PLA) and poly (glycolic acid) (PGA). Biomaterials can be used to increase tissue functions and also treat diseases and injuries. Therefore, biomaterials are in contact with biological systems. When biomaterials come into contact with blood, the first thing that happens is the adsorption of blood proteins in the solid (polymer) - liquid (blood) interface. It is crucial to understand the mechanism of protein adsorption and control protein adsorption process onto polymer surfaces in order to characterize polymeric biomaterials for different biomedical applications. Since protein adsorption directly affects biocompatibility, biological interaction and material performance in vivo, it is important to characterize protein adsorption onto polymer surfaces. There is a number of different factors affecting protein adsorption process, and controlling this process is cruicial since the properties of protein, solution and adsorbent surface have a synergistic effect on protein adsorption. Protein properties including the size, flexibility and hydrophilicity affect protein adsorption rate onto polymer surfaces. In addition to chemical, geometric, and electrical properties of the polymer surface, properties of the solution such as pH, temperature, ionic strength, chemical composition, and concentration also affect the protein adsorption rate.
In this study, polyurethane (PU), a biocompatible polymer, was studied, and the effects of roughness and crystallinity on fibrinogen adsorption were determined by both experimental and mathematical modeling and simulation methods. In the experimental study, PU film synthesis with different surface properties were performed, and its properties were characterized. Accordingly, adsorption of fibrinogen protein onto PU surfaces was investigated. Due to the favorable physical and chemical properties and biocompatibility of PU, it is often used as a biomaterial in biomedical applications. For example, PU can be designed to provide structural scaffolding in tissue engineering. Pores in the structure of PU could provide cellular filtration and food transport to derive formation of new blood vessels. PU materials can be also used as a catheter or cardiac valve in the biomedical applications.
There are two methods to synthesize PU, and these are one shot and prepolymer methods. If the one shot method is preferred, all reactants are placed in the reaction medium, and required reaction conditions like temperature are adjusted to complete PU synthesis reaction. When it is desired to synthesize using the prepolymer method, the diol and the diisocyanate are reacted to form PU prepolymer. Then, a chain extender is added to the reaction mixture to form PU polymers. Prepolymer method is generally preferred to control the PU structure. The properties of the PU mainly depend on the content of the reactive groups, the degree of cross-linking in the structure and the degree of entanglement of the reacting monomers. Petroleum-based polyols and vegetable oils can be used in PU synthesis. However, the use of renewable raw materials such as vegetable oils is preferred due to environmental concerns and economic reasons in the synthesis. Moreover, low toxicity, high purity and cheapness of vegetable oils increase their preference. Soybean oil, canola oil, palm oil, sunflower oil, corn oil and castor oil can be used for this purpose. In this study, castor oil is selected for PU synthesis. Castor oil provides better chemical and mechanical properties to PU since castor oil has amorphous structure and a hydrophobic character. Moreover, castor oil acts as a crosslinker in PU synthesis. Although the activity of aromatic isocyanates is higher than aliphatic isocyanates, usage of aromatic diisocyanates are not preferred in PU synthesis since aromatic diisocyanate compounds have toxic effect in vivo due to aromatic structures. Hexamethylene diisocyanate, an aliphatic diisocyanate, is used in the PU synthesis as isocyanate source. In the PU synthesis, 1,4-butane diol (BDO) is used as chain extender. The selected reactants determine the properties of the PU, so the reactants used in the synthesis cause the formation of hard and soft regions in PU. Short chain diols such as BDO and diisocyanates form the hard region of PU, while long chain diols such as polyethyleneglycol (PEG) form the soft region. The hard region determines the rigidity and tear strength of PU, while the soft region determines flexibility, softness, and resistance of PU to low temperatures.
Major blood proteins are albumin, hemoglobin, gamma globulin, fibrinogen and ferritin. In the previous study, albumin adsorption was investigated on various PU surfaces. Albumin is smaller than fibrinogen, and it has isotropic structure. In this study, the effect of anisotropic and flexible structure of fibrinogen on adsorption is investigated; hence, adsorption analysis of fibrinogen on various PU surfaces is conducted. When biomaterials are transplanted into the body, first they come into contact with blood, primarily with the blood proteins including fibrinogen. Fibrinogen adsorbs onto the surface of biomaterials and then catalyzed by thrombin in order to form fibrin, which can then initiate clot formation on the surface of the material. Therefore, fibrinogen adsorption onto PU surfaces needs to be characterized in order to optimize the surface properties of PU for biomedical applications.
The characterizations of synthesized PU films were made by FTIR, DSC, water contact angle, swelling and AFM analysis. The steps of polymerization reaction were observed by FTIR analysis.
In the beginning of the reaction, OH- strain peak and free isocyanate peak were observed in the FTIR spectrum. When the reaction was complete, OH- strain peak and the free isocyanate peak were disappeared, and the characteristic urethane peak was observed. Thermodynamic properties of materials can be determined by thermal analysis using DSC. DSC analysis shows that as the amount of cross-link in the films increases, the movement of the polymer chains becomes more difficult, and glass transition temperature (Tg) of the PEG-free PU film is getting higher. Water contact angle and swelling analysis give information on hydrophilicity of PU surfaces. When the PEG content of PU film increases, the PU film hydrophilicity increases.
Accordingly, the surface properties of PU films are determined by AFM analysis. The surface roughness and roughness width of PU films were obtained by AFM analysis. It was found that the surface roughness tends to increase as the amount of PEG and the molecular weight of PEG increases. As a result of experimental studies, it was shown that the hydrophilicity, crystallinity, and surface roughness of the polymer determine the fibrinogen adsorption rate on PU surfaces. The surface roughness controls the fibrinogen adsorption onto PU-100 and PU-400 coded films. The surface roughness and film hydrophilicity control the adsorption onto PU-1500 coded films, while the main parameter controlling the adsorption is the film hydrophilicity for PU-3000 coded films. As the molecular weight of PEG increased, the effect of surface roughness on the adsorption decreased and the effect of hydrophilicity became dominant. As a result of the experimental study, it was shown that the factor controlling the fibrinogen adsorption was transmitted from the surface roughness to the hydrophilicity by increasing the molecular weight of PEG.
Since fibrinogen has an anisotropic and flexible structure, the simulation study was started by determining the flexible regions and hinge movements of the fibrinogen using the HingeProt internet server. Gaussian network model was used to determine the flexible parts of the fibrinogen and hinge movements. Fibrinogen makes the bending and twisting movements. The flexible parts are located in the middle and two ends of the fibrinogen, so the fibrinogen was modeled as three nodules connected to each other with springs. After the determination of the fibrinogen model, the effects of surface roughness and crystallinity on adsorption were investigated by Brownian dynamics simulation. In order to investigate the effect of surface crystallinity on protein adsorption, two types of flat surfaces in which the adsorption regions were arranged in random order were modeled. Three types of rough surfaces were modeled in a random distribution of adsorption regions with different roughness and roughness depths, and the effect of surface roughness on fibrinogen adsorption was analyzed by Brownian simulation method. As a result of the simulation study, it was shown that crystallinity and surface roughness due to the steric hindrance of fibrinogen determine the fibrinogen adsorption rate onto PU surfaces. When the results of the experimental study were compared with the results of the simulation study, it was concluded that fibrinogen adsorption increased onto PU surfaces with the increase of surface roughness.