Kanser tedavisinde ilaç taşıma amacıyla kullanılacak karbon nanotüplerin fonksiyonelleştirilmesi
Functionalization of carbon nanotubes to be used for drug delivery in cancer treatment
Yazar: YEŞİM YENİYURT
Danışman: PROF. DR. FATMA SENİHA GÜNER
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:
Dünyada ölümlerin önde gelen nedenlerinden biri olan kanserin tedavisi için kullanılan yöntemler sistemik etki göstermektedir. Biyomedikal alandaki gelişmeler sayesinde gelişen ilaç taşıma sistemleri ilacın tümörlü bölgeye nüfuzunu arttırabilmekte ve nano boyuttaki dağıtım sistemleriyle dokulara doğrudan iletilebilmektedir. Böylece sistemik etki yerine lokal etki sağlayabilmektedir. 

Yapılan bu çalışmada, ilaç taşıyıcısı olarak kullanılabilecek yeni bir nanomalzeme geliştirilmiştir. Nanomalzemenin iskeleti, fizikokimyasal özelliklerinin diğer nanomalzemelere göre daha üstün olması sebebiyle karbon nanotüp (KNT) olarak seçilmiştir. KNT'lerin çözünürlüklerinin az olması kullanımını sınırlayıcı bir özelliktir. Hem bu problemi çözmek hem de retiküloendotelyal sistem elemanları tarafından KNT'lerin yabancı madde olarak algılanmasını önlemek ve böylece ilaç yüklü KNT'lerin kanda kalma süresini artırmak amacıyla yüzeyleri hidrofilik ve biyouyumlu bir polimer olan polietilen glikol (PEG) ile kaplanmıştır. KNT yüzeyinin hidrofobik olması nedeniyle yüzeyin hidrofilik bir polimerle kaplanması işlemi birkaç basamakta gerçekleştirilmiştir. İlk olarak PEG zincirine piren gibi aromatik gruplar içeren 9-florenilmetil kloroformat (Fmoc) aminoasitleri bağlanmış, daha sonra uzun PEG zincirlerinin ᴨ-ᴨ etkileşimleri sayesinde tek duvarlı KNT (TDKNT) duvarına tutturulması sağlanmıştır. Çalışmada, Fmoc aminoasitlerine monte edilmiş farklı molekül ağırlığındaki PEG (Ma=2000, 5000 ve 12000 g mol-1) ile KNT yüzeyinin non-kovalent fonksiyonelleştirilmesi başarıyla gerçekleştirilmiştir. Sentezlenen nihai üründe Fmoc-PEG zincirlerinin TDKNT'lere bağlanmasını izlemek amacıyla floresans spektroskopisi, bağlanma verimini saptamak amacıyla da termogravimetrik analiz yöntemi kullanılmıştır. En yüksek biyouyumluluk gösteren nanotaşıyıcı kanser tedavisinde kullanılmak üzere ilaç yükleme ve kontrollü salım çalışmaları için seçilecektir.

Abstract:
Methods used for the treatment of cancer, one of the leading causes of death in the world, show a systemic effect. The drug delivery systems improved by the developments in the biomedical field can increase the penetration of the drug into the tumor region and can be transmitted directly to the tissues by means of nano-sized delivery systems. Thus, it can provide local effect instead of systemic effect. 
In this study, a new nanomaterial which can be used as a drug carrier has been developed. Nanomaterial skeleton was chosen as carbon nanotube (CNT) because its physicochemical properties were superior to other nanomaterials. The low solubility of CNTs is a limiting feature. To solve this problem, the surfaces of the CNTs are coated with polyethylene glycol (PEG), a hydrophilic and biocompatible polymer. This method prevents the detection of CNTs as foreign substances by reticuloendothelial system elements and increases the residence time of drug-loaded CNTs in the blood. Because the surface of the CNT is hydrophobic, the coating of the surface with a hydrophilic polymer is carried out in several steps. Firstly, 9-fluorenylmethyl chloroformate (Fmoc) amino acids containing pyrene-like aromatic groups were attached to the PEG chain, and then PEG chains were attached to the single wall carbon nanotubes (SWNT) wall due to π-π interactions. 
In this study, non-covalent functionalization of the SWNT surface with different molecular weight PEG (Mw = 2000, 5000 and 12000 g mol-1) mounted on Fmoc amino acids was successfully. Non-covalent functionalization was performed in order to improve biocompatibility and increase water solubility of CNTs and two different Fmoc amino acids such as glycine (Gly) and tryptophane (Trp) bearing PEG were attached to the CNTs surface. Fmoc amino acids are used for direct CNT immobilization. In addition, it is known that Fmoc amino acids can interact through the π-π bond, since such molecules can self-assembly with nanostructures. This system designed for drug delivery was synthesized in three steps. Primarily, SWNTs were synthesized using chemical vapor deposition method to form the skeleton of the system.  Then, complexes were used to modify the SWNTs. The Fmoc amino acids and PEGs are linked together via the Sterlich Esterification mechanism. In the last step, f-PEG (Fmoc-Gly-PEG and Fmoc-Trp-PEG) prepared by reacting PEG and SWNTs were treated in ultrasonic bath to mount the complexes on the nanotube wall.  Based on the studies in the literature, it has been demonstrated that PEG/SWNTs carrying two different Fmoc-aa molecules have the necessary structural strength. Stability, binding efficiency and suspending properties of Fmoc-PEG coated SWNTs were investigated by characterization studies.
 1H Nuclear Magnetic Resonance Spectroscopy (NMR) were used to monitor the synthesis of Fmoc-PEG complexes, the first step of the synthesis. The characteristic peaks of Fmoc-Gly-OH and Fmoc-Trp-OH indicate that these structures are successfully linked to PEG.
Fourier-transform infrared (FT-IR) spectra were examined to support the 1 H NMR spectrum. When the PEG and f-PEG spectras are compared, the characteristic peaks of the Fmoc groups at 1770 cm-1 clearly demonstrate binding.
In the second step of the synthesis, PEGs of different molecular weight to which the Fmoc-amino acids are bound were mounted to SWNTs. In this step, binding was determined by fluorescence spectrophotometer and the amount of binding was determined by thermogravimetric analysis (TGA).
For each sample, two different fluorescence spectra were obtained that characterize Fmoc-PEG and Fmoc-PEG / SWNTs. It has been reported in the literature that the π-π bond between aromatic rings may cause the damping of the fluorescence spectrum in energy transfer. In addition, it was predicted that Fmoc-Trp-OH would bind more to SWNTs surface due to aromatic rings containing tryptophan amino acid. In order to confirm it and to eliminate the chain length effect of PEG, the amount of Fmoc amino acids bound to SWNTs was determined and TGA analysis was performed as described in the experimental part. Both Fmoc-Gly-OH and Fmoc-Trp-OH were found to bind to the surface of SWNTs. As expected, the amount of mass loss for Fmoc-Trp-OH is higher than Fmoc-Gly-OH. Aromatic fluorenyl rings are capable of binding to the surface of SWNTs through π – π interactions in both Fmoc-Gly-OH and Fmoc-Trp-OH. The presence of the aromatic tryptophan ring in Fmoc-Trp-OH promoted binding to SWNT walls.
In order to examine the effect of the amount of PEG on SWNTs surface binding efficiency, PEG5000 was taken as reference and experiments were performed using different amounts of PEG. The binding efficiencies of Fmoc-Gly-PEG5000 and Fmoc-Trp-PEG5000 to the SWNTs surface were monitored for different molar masses. Increasing the amount of PEG had a positive effect on both glycine and tryptophan complexes and increased binding efficiency.
Due to the presence of aromatic fluorenyl rings, Fmoc group was easily mounted on SWNT walls and it was determined by using TGA analysis that tryptophan was bound to SWNTs more homogeneously than glycine by aromatic rings. Glycine and tryptophan showed completely different tendencies in TGA analysis to determine the binding amount of complexes with PEG. In the Fmoc-Gly-PEG/SWNTs thermograms, the percentage of mass loss increased as the molecular weight of PEG increased, and the highest yield was obtained from PEG2000. In the Fmoc-Trp-PEG/SWNTs thermograms, the percentage of mass loss decreased with increasing molecular weight of PEG.
To determine the dispersion behavior of SWNTs modified by Fmoc amino acids, CNT dispersions were prepared and obtained by non-covalent modifications using PEG with aromatic functionality mounted on nanotube surfaces by π-π interactions. Additional interactions between surface-bound and non-bound Fmoc amino acids also occur through π-π stacking and hydrogen bonding; this may contribute to the overall formation of a stable nanotube dispersion in aqueous medium. The dispersion of SWNTs in water was examined in 0, 1, 3 and 5 hours and Fmoc-Trp-OH was found to provide the best dispersing property.
The binding of Fmoc amino acids containing PEG chains to the CNT wall combines the strengths of the PEG chain and CNTs; A nanocarrier is synthesized with a blood compatible, stable cell structure and good optical properties. The synthesized product will be a nanotransmitter capable of delivering targeted drug delivery and release, minimizing the damage / side effects, in line with the need for alternative methods of cancer treatment.
In studies in the literature, it is determined that paclitaxel molecule is loaded to Fmoc amino acids complexes for cancer treatment. In the following steps, the loading efficiency of the drug suitable for the nanomaterials synthesized in this study will be investigated. The resulting nanocarrier is expected to have high drug carrying capacity thanks to Fmoc groups. In addition, in order to understand the biological behavior of f-SWNTs, its toxic effect on the cancer cell should be determined. In vitro cytotoxicity tests will be performed in cell culture in order to evaluate this material, whose drug profile and toxic profile has been investigated. The nanomaterials obtained in this way, with their advanced properties, promise great future as pharmaceutical transportation vehicles.