Çinko nanotanecik içeren polimer nanokompozit malzeme üretimi ve karakterizasyonu
Fabrication and characterization of polymer nanocomposite materials incorporated zno nanoparticles
Yazar: ALEV AKBAŞ
Danışman: PROF. DR. SADRİYE KÜÇÜKBAYRAK OSKAY
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Mühendisliği Ana Bilim Dalı / Kimya Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2018
Konu: Kimya Mühendisliği = Chemical Engineering

Özet:
Nanoteknoloji, en az bir boyutu 1-100 nm arasında değişen nanomalzemelerin sağladığı fiziksel, kimyasal ve elektriksel özellikleri kullanarak yeni özelliklere ve işlevlere sahip malzemelerin üretimini ve geliştirilmesini hedefleyen çok yönlü bir çalışma alanıdır. Nanomalzemeler nanoboyuta inildikçe artan yüzey / hacim oranının sağladığı özellikler ile elektronik, biyomedikal, gıda ve enerji gibi alanlarda kullanılmaktadır.
Nanoteknolojinin önemli uygulama alanlarından biri olan doku mühendisliği alanında yer alan yara örtüsü ile zarar görmüş hücrelerin ve dokuların onarılması için yara bölgesine hücrelerin göçünü, çoğalmasını ve farklılaşmasını sağlamak için ekstraselüler matriks (ECM) görevi görebilecek yapıların geliştirilmesi amaçlanır. Yara örtüsünün kullanım amaçları yaraların iyileşmesi için gerekli olan nemi ve gaz geçirgenliğini sağlamak, enfeksiyonu önlemek veya kontrol altında tutmak, yara eksudasını temizlemek ve ağrının en aza indirilmesini sağlamaktır. Sentetik (PCL, PLA vb.) ve doğal biyopolimerler (kitosan, jelatin, kolajen vb.) ile çeşitli lif üretim yöntemleri kullanılarak genellikle nanolif matriks yapısında oluşturulan yara örtüleri içerisine yara iyileşmesini hızlandırmak, ağrıyı azaltmak, tedavi etmek ve antibakteriyel özellik kazandırmak için ilaçlar, büyüme faktörleri veya nanotanecikler yerleştirilerek zarar görmüş dokunun onarılması sağlanmaktadır.
Biyopolimer çeşidi olan polikaprolakton (PCL) toksik olmaması, mekanik özelliklerinin iyi olması, biyouyumluluk ve biyolojik bozunabilirlik özelliklerinin getirdiği avantajlar sayesinde biyomedikal alanda ilaç taşıyıcı sistemlerde ve doku mühendisliği uygulamalarında iskele ve yara örtüsü olarak kullanılabilmektedir.
Aktif yara örtüsü çalışmalarında yara iyileşmesini hızlandırmak için ZnO nanotaneciği kullanılmaktadır. Vücudumuzda var olan elementlerden biri olan ve ekstraselüler matriks oluşumunu destekleyen metaloproteinlerin kofaktörü olan çinko ile yara iyileşme süresi kısaltılabilmektedir. Çinko kaynağı olarak kullanılabilen ZnO nanotaneciklerinin ROS üretimine neden olarak, ya da bakteri hücre duvarına zarar vererek yara bölgesinde antibakteriyel özellik gösterdiği ve yara bölgesine keratinosit ve fibroblast hücrelerinin göçünü ve çeşitlenmesini sağlayarak, yara iyileşmesini hızlandırdığı belirtilmiştir.
Genellikle vitamin A ve E, cilt için bölgesel kremler, losyonlar veya merhemler de kullanılan cildin yaşlanmasını geciktiren ve antioksidan özellik sağlayan terapötik ajanlardır. ZnO nanotanecikleri gibi vitamin A ve vitamin E'nin de yara iyileştirmede olumlu sonuçlar sağladığı yapılan çalışmalar ile görülmüştür. Vitamin E yara bölgesinde antioksidan özellik sağlamakta ve vitamin A ise kolajen sentezini desteklemektedir.
Yapılan çalışmaların incelenmesi sonucu bu çalışmada, mikrodalga destekli hidrotermal yöntem ile ZnO nanotaneciğinin üretimi ve elektrospinning yöntemi ile ZnO nanotaneciği, vitamin E ve A katkılı PCL nanokompozit lif yara örtüsünün üretimi amaçlanmıştır.
Deneysel çalışmaların birinci bölümünde; Zn(NO3)2.6H2O, NaOH, stabilize edici ajan olarak nişasta ve çözücü olarak su kullanılarak ZnO nanotaneciğinin üretimi mikrodalga destekli hidrotermal yöntem ile gerçekleştirilmiştir. Bu bölümde nanotaneciğin boyutuna ve morfolojisine Zn(NO3)2.6H2O ve NaOH derişimleri, pH, mikrodalga gücü ve mikrodalga uygulama süresinin etkisi incelenerek nanokompozit lif yapılarının üretiminde kullanılabilecek ideal nanotanecik üretimi gerçekleştirilmiştir.
Deneysel çalışmaların ikinci bölümünde; asetik asit-formik asit çözücü karışımı kullanılarak saf PCL, çeşitli oranlarda ZnO nanotaneciği ve vitamin E ve A içeren ZnO/PCL, VİT/PCL, ZnO/VİT/PCL nanoliflerinin üretimi elektrospinning prosesi ile gerçekleştirilmiştir.
Üretimi gerçekleştirilen nanotaneciklerin ve nanoliflerin karakterizasyonu SEM, FTIR ve XRD kullanılarak gerçekleştirilmiştir. ZnO/PCL, VİT/PCL, ZnO/VİT/PCL nanokompozit lifleri için belli bir zaman periyodunda çinko iyonu ve vitamin salım testleri uygulanmıştır. Elde edilen liflere antibakteriyel aktivitelerini incelemek için antimikrobiyal testler uygulanmıştır.
Sentezi gerçekleştirilen ZnO nanotaneciklerinin morfolojisi ve tanecik boyut dağılımı üzerinde Zn(NO3)2.6H2O ve NaOH derişimleri, pH, mikrodalga gücü ve mikrodalga uygulama süresi parametrelerinin etkileri olduğu görülmüştür. Küresel formda elde edilen nanotaneciklerin, ortalama tanecik boyutu; Zn(NO3)2.6H2O ve NaOH derişimi ve çözelti pH artışı ile büyümüş, mikrodalga uygulama süresinin ve gücünün artması ile küçülmüştür.
Nanokompozit lif yapılarında, saf PCL nanoliflerine ZnO nanotaneciklerinin ilavesi ile ortalama lif çaplarında küçülme ve vitaminlerin ilavesi ile ortalama lif çaplarında büyüme görülmüştür. Nanokompozit yapıdan çinko iyonu ve vitamin salımının gerçekleştiği belirlenmiştir. Ayrıca, yüksek ZnO derişimi içeren nanokompozit lif yapıların Escherichia coli ve Staphylococcus aureus bakterilerine karşı antibakteriyel özellik sağladığı görülmüştür.
Sonuç olarak, üretimi gerçekleştirilen nanokompozit lif yapılarının yara örtüsü olarak kullanılma potansiyeline sahip olduğu saptanmıştır.

Abstract:
Nanotechnology is a versatile field to develop, to fabricate and to design materials and processes with nanosize. According to the International Organization for Standardization (ISO), the prefix nano refers to a size ranging approximately from 1 to 100 nm. Nanotechnology is interested in nanomaterials that have considerable biological, chemical, magnetic and physical properties due to their nanoscale size. Nanomaterials are the building blocks of nanotechnology and is a term used to describe materials having at least one dimension 100 nm or less. Nanomaterials can be classified according to their dimensions and chemical structures. According to their dimensions, nanomaterials are composed of four groups that are zero (0D), one (1D), two (2D) and three dimensional (3D). Nanomaterials can also be classified based on chemical structure as metal-based, metal-oxide based, carbon-based, dendrimers, quantum dots, and polymeric materials. Nanotechnology have wide range of applications in the field of electronics, agriculture, food industry, biomedical, cosmetics, energy and environment.
Production of nanomaterials are generally classified in two main approaches as bottom-up approach and the top-down approach. The bottom-up approach refers to the production of nanomaterials by physical, chemical or biological methods by using atomic or molecular level material. On the other hand, the top-down approach is based on the separation of large pieces of material at the macroscopic level followed by mechanical and / or chemical treatments to obtain the desired nanomaterials.
Nanoparticle production by the microwave method has advantages compared to other methods because it is cheap and ensures short reaction time, homogenous distribution of heat for the particles, achieving the small particle size with narrow size distribution, high purity product and helps to reduce agglomeration. Solution parameters such as concentration and pH of the solution and microwave conditions such as microwave power and irradiation time which are important parameters affecting the size and distribution of the produced nanoparticle in the microwave method.
Nanofibers are one-dimensional (1D) nanomaterials with a "high surface-to-volume ratio" due to nanometer scale and this property helps to increase interactions between the nanofibers and targeted substrates in many application fields especially in biomedical applications. They can be fabricated by various methods which are electrospinning, self assembly or template-assisted growth. Electrospinning method has many advantages in nanofiber production. It can be applied to fabricate fibers having sizes between nanometers and micrometers. Continuous and uniform, structures with controllable porosity formation can be obtained by applying an electrical force. The structure and size of the fibers produced by the electrospinning method are influenced by the solution (eg viscosity, concentration, etc.), the process (eg voltage, feed rate etc.) and the environmental parameters (eg humidity and temperature). The nanofiber mats with high porosity, gas permeability and high surface area produced by the electrospinning process can be used in a variety of biomedical applications such as wound dressings, drug delivery systems and tissue scaffolds. Nanofiber structures play an important role in promoting the proliferation and migration of cells in tissue engineering applications with structural similarity, which is an extracellular matrix (ECM).
Biopolymers can be defined as polymers obtained from renewable sources or biodegradable polymers derived from biological and fossil fuel sources. Biopolymers are mostly used in medical, agriculture and food fields. Polycaprolactone (PCL) is a biodegradable aliphatic polyester derived from fossil fuel sources. Due to its non-toxicity, good mechanical properties, biocompatibility and biodegradability, it can be used in drug delivery systems and tissue engineering applications and wound dressing applications in biomedical field.
Tissue engineering is one of the key applications of nanotechnology focuses on the development of structures that can serve as ECM to repair damaged tissues, cells or organs. ECM can help to achieve biomimetic migration and proliferation of cells at the applied damaged zone using advantages of nanosize. Wound dressings which are one of the studying subject of tissue engineering are structures that act as a physical barrier between the wound and its surroundings. Wound dressings are classified into two groups which are passive and active wound dressings. Passive wound dressings include traditional dressings such as cotton gauzes and modern wound dressings such as foam and film forms of alginate, chitosan and polyurethane. This kind of wound dressings are insufficient to meet the ideal wound dressing properties. Active wound dressings have being developed in order to overcome this problems. Active wound dressing are fabricated from synthetic (PCL, PLA, PVA etc.) and natural biopolymers (chitosan, gelatin, alginate, etc.) which are impregnated with antimicrobial drugs, collagen, enzymes, ZnO, silver nanoparticles, growth factors and vitamins to accelerate wound healing. The ideal wound dressing should provide the moisture environment and oxygen permeability necessary for wound healing, prevent or control microbial biofilms, absorbtion the exudate from an open wound, biocompatibility for skin and biodegradability.
Vitamins A and E are usually used for topical creams, lotions or ointments for the skin. These are therapeutic agents that delay the aging of the skin and provide antioxidant properties. Vitamin A and vitamin E have also been shown to provide positive results in wound healing. Vitamin E provides antioxidant properties and vitamin A supports collagen synthesis in the wound area.
Zinc oxide (ZnO) is a unique material with a wide band gap of 3.37 eV and a large excitation binding energy (60 meV). ZnO is currently listed as a 'generally recognized as safe' (GRAS) material by the Food and Drug Administration and is used as a food additive. ZnO NPs are one of the best appropriate source for wound healing applications by enhancing re-epithelialization, reducing inflammation, inhibiting bacterial growth by generating reactive oxygen species (ROS) and releasing zinc ions. ZnO NPs can also improve cell adhesion, proliferation and cell migration through growth factor mediated pathways by causing (ROS) production. ZnO acts as a zinc source. Zinc is one of the important elements found in the human body. It is involved as a cofactor in various transcription factors and enzyme systems, including zinc-dependent matrix metalloproteinases and zinc finger proteins. Zinc finger proteins are one of the transcription factor family and they specifically bind to DNA and activate the transcription of growth factors. Matrix metalloproteinases (MMPs) which contain a zinc ion in the active site improve autodebridement and keratinocyte migration during wound repair. These enzymes play an essential role in physiological states such as tissue remodeling, morphogenesis and wound healing and they are capable of degrading extracellular matrix and basal membrane components. ECM is a complex and dynamic formation and includes proteins and proteoglycans. It provides structural support to the organism, cell proliferation, differentiation and adhesion with migration. Metalloproteinases are all proenzymes secreted from various connective tissue cells such as fibroblasts, osteoblasts, endothelial cells, macrophages, neutrophils.
This study consists of two parts which are synthesis of ZnO nanoparticles by microwave method and fabrication of PCL nanocomposite fibers doped with ZnO nanoparticles, vitamin A and E by electrospinning method.
In the first part of the study, ZnO nanoparticles were produced by microwave assisted hydrothermal method. Soluble starch, Zn(NO3)2.6H2O, NaOH and water used as solvent in the synthesis of nanoparticles. Production of nanoparticle in spherical form was aimed due to nanoparticles in the spherical form have a large surface area / volume ratio, thus the interaction of the nanoparticle with the target surface is increased. The use of water as a solvent causes agglomeration of nanoparticles. Soluble starch was used to reduce the agglomeration and to produce nanoparticles in spherical shape. Preliminary experimental studies have been carried out with synthesis conditions such as different concentrations of Zn(NO3)2.6H2O and NaOH, various pH, microwave power and time to obtain nanoparticles with homogeneous morphology, high efficiency and ideal size. According to the results obtained by SEM analysis and Image J program, increase in pH and concentrations of Zn(NO3)2.6H2O and NaOH lead to an increase in the average size distribution of nanoparticles. On the other hand, increase in the microwave power and the microwave irridation time resulted to reduction in the average size distribution of the nanoparticles. The ideal nanoparticle size distribution was obtained as 233 ± 48 nm and nanoparticles were in spherical form using 1 M Zn(NO3)2.6H2O aqueous solution, 2 M NaOH aqueous solution, 600 W microwave power and 2 minutes irradiation time at pH = 7. XRD analysis of the nanoparticles revealed formation of ZnO nanoparticles. Also, FT-IR analysis of ZnO nanoparticles showed the presence of starch in the structure. According to the XRD analysis, the synthesis of the ZnO nanoparticles were successfully accomplished.
In the second part of the study, neat PCL, ZnO / PCL containing ZnO nanoparticles, VIT / PCL containing vitamins A and E and ZnO / VIT / PCL containing both vitamins and nanoparticles nanocomposite fiber structures were produced by electrospinning method. The addition of ZnO nanoparticles and vitamins into neat PCL nanofibers causes to change the fiber morphology and average fiber diameter. It was observed that the fiber diameter increased with the increasing amount of ZnO nanoparticles and there was a decrease in the homogeneous distribution of the fibers due to aglomeration of ZnO nanoparticles. Furthermore, the addition of vitamin A and vitamin E lead to increase the average nanofiber diameter. The presence of the ZnO nanoparticles in the ZnO / PCL nanofibers was proved by XRD and EDX analyzes. In the FTIR analysis of the produced nanofibers, the characteristic peaks of the PCL polymer were observed. In vitro release test of zinc ion from ZnO/PCL nanocomposites showed slow release behavior for a certain time in PBS medium. In addition, the amount of released zinc ion is below the toxic values given in the literature.
In vitro release test of vitamins from VIT/PCL and ZnO/VIT/PCL nanocomposites showed slow behavior for a certain time in PBS medium. At the end of the third day, released amounts of vitamins were below compared to literature values. It may be due to the slow rate of degradability the PCL polymer.
The antimicrobial test with nanocomposite fibers showed that 5% ZnO / PCL, 10% ZnO / PCL, 15% ZnO / PCL, 10% ZnO / VIT / PCL and 15% ZnO / VIT / PCL nanocomposite fibers were antibacterial against E. coli. In addition, 10% ZnO / PCL, 15% ZnO / PCL and 15% ZnO / VIT / PCL nanocomposite fibers were antibacterial against S. aureus. ZnO / PCL nanofibers were found to be more effective compared to the inhibition zone diameters of ZnO / PCL and ZnO / VIT / PCL nanocomposite fibers which contain the same amount of ZnO. It can be assumed that vitamin A has a positive effect on bacterial growth.
15% ZnO / PCL and 15% ZnO / VIT / PCL nanocomposite structures showed antibacterial properties, high vitamin and zinc ion emission levels and potency to be used as wound dressings.
Fabricated nanocomposite materials can be a potential candidate to be used in wound dressing applications due to their ability to release ZnO ions and vitamins and antimicrobial properties.