Kitosan/ZnFe2O4 nanokompozit malzemesi hazırlanması, karakterizasyonu ve kanser ilacı yüklenen malzemeden ilaç salımının incelenmesi
Preparation, characterization and examination of drug release from cancer drug loaded nanocomposite chitosan/ZnFe2O4 material
Yazar: MERVE ECE TEMELKURAN
Danışman: PROF. DR. FATMA BEDİA BERKER ; ÖĞR. GÖR. ZEYNEP KALAYCIOĞLU
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Kimya Ana Bilim Dalı / Kimya Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Biyokimya = Biochemistry ; Biyoloji = Biology ; Kimya = Chemistry
Dizin: Kitosan = Chitosan ; Nanopartiküller = Nanoparticles ; Çinko ferrit = Zinc ferrite ; İlaç dağıtım sistemleri = Drug delivery systems ; İlaç salınımı =   ; İlaç taşıyıcılar = Drug carriers

Özet:
Mevcut farmasötik teknolojide, antitümör ilaçların hedefe yönelik iletimi ve uzun süreli salım için taşıyıcıların tasarlanması büyük önem kazanmıştır. İlaçlar içerisinde mevcut hastalığı tedaviye yönelik bulunan etken maddeleri, vücutta hastalığın bulunduğu yer dışında birçok yere ulaşarak orada birikebilir. Kontrolsüz uygulanan ilaçlar sonucunda vücutta birçok yan etki oluşumu görülebilir. Bu nedenle etken maddeye ve hastalığa göre tasarlanmış olan ilaç taşıyıcıları ve kontrollü ilaç salım sistemleri bulunmaktadır.
Pek çok taşıyıcı arasında hidrojel polisakkarit nanoparçacıkları son zamanlarda giderek daha fazla ilgi görmektedir. Kitosan biyouyumlu, biyolojik olarak parçalanabilen ve bakteriyostatik olarak bilinen doğal bir polimerdir. Aynı zamanda uygun maliyetlidir ve yüksek bir adsorpsiyon potansiyeli sergiler. Kitosanın ilaç salımı üzerine uygulamaları filmler, hidrojeller, lifler, mikro/nanokapsüller ve mikro/nanoparçacıkları içermektedir. 
Manyetik ayrılabilirlik tıbbi uygulamaların genişletilmesinde önemli bir rol oynadığından son zamanlarda ilaç taşıyıcı sistemlerde manyetik nanoparçacıklar kullanılmaktadır. Bu sayede normal hücrelere göre manyetik çekim özelliği farklı olan tümör hücrelerine spesifik ilaç iletimi sağlanmış olur.
Bu araştırmanın temel amacı, bir kanser ilacı olan imatinibin kontrollü salımı için kitosan ile modifiye edilmiş çinko ferrit oksit (ZnFe2O4) nanokompozit malzeme geliştirilmesidir. ZnFe2O4, paramanyetik ve antibakteriyel bir nanometal oksittir. 
Hedeflenen nanokompozit malzeme iyonik jelleşme tekniği ile hazırlanmıştır. Daha sonra elde edilen ZnFe2O4-kitosan nanokompozit malzeme ticari ismi "Glivec"  olan bir tirozin kinaz inhibitörü imatinib ile yüklenmiştir. İmatinib yüklü ZnFe2O4-kitosan nanoparçacıkları, ilaç enkapsülasyon etkinliği ve in vitro ilaç salımını araştırmak için kullanılmıştır. Enkapsülasyon verimliliği %77,8 olarak bulunmuştur. ZnFe2O4 nanoparçacıkların ilaç enkapsülasyon etkinliği üzerindeki etkisinin incelenmesi için ZnFe2O4 içermeyen kitosan-nanoparçacıklar hazırlanmış ve kitosan-nanoparçacıkların ilaç enkapsülasyon etkinliği ise %36,0 olarak bulunmuştur. ZnFe2O4 katkısının ilacın enkapsülasyon etkinliğini önemli ölçüde artırdığı görülmektedir. Nanoparçacıkların karakterizasyonu fourier dönüşümlü kızılötesi spektroskopisi (FTIR) ve taramalı elektron mikroskobu (SEM) analiz teknikleri ile gerçekleştirilmiştir. İlaç içeren kompozitin ilaç salım profili 37 °C ve pH 7.4 olan fosfat tamponlu tuz çözeltisinde incelenmiştir.
Bu tez çalışmasının, kanser tedavi yöntemlerinin ve ilaç salım tekniklerinin geliştirilmesine yönelik araştırmalara katkı sağlaması beklenmektedir.

Abstract:
In the current pharmaceutical technology, it has been gained a huge importance to design carriers for targeted delivery of antitumor drugs and prolonged release in tumor cells. Since the active ingredient of the drug product could travel anywhere in the body and could accumulate other than it is sposed to be, controlled drug released takes an important role for the positive efficiency of the treatments for the diseases. Likewise, controlled drug release is extremely important because of the high or unbalanced dosage exposure also leads negative results for the patient during the treatment. Therefore, special carriers are used for active ingredients to be taken to the right and exect place inside the body with optimum amount of relase.
Nanotechnology is the creation of new structures by directing the substance at atomic and molecular levels and changing its properties. The materials used in nanotechnology are generally 1-100 nanometer (nm). Nanotechnology has created many radical effects among various disciplines and is one of the most important application areas. For medical purposes such as early detection of pathological processes, precautions and targeted treatments, it can be defined as the use of organic or synthesized nano-scale materials, chemical, physical, electrical, optical and biological properties. The most important area of use of nanotechnology in medicine is cancer diagnosis and treatment, that is, often referred to as 'teranostic or teraghnostic'. Nanoparticles overcome biological barriers and facilitate the follow -up of diagnosis, treatment, disease and treatment response. For this purpose, nanotechnology is used in many studies and researches, especially tumors.
Among many carrier, hydrogel polysaccharide nanoparticles (NPs) have been increasingly interested recently. Chitosan (CS) is a natural polymer which known as biocompatible, biodegradable, and bacteriostatic.  It is also cost-effective and exhibits a high absorption potential. Chitosan is widely used in many fields of studies in industrial and academic fields due to its lack of toxic, not toxic, biocyumluolism and naturally obtained. Chitosan is found in the outdoor skeletons of shellfish such as crab and shrimp, butterflies' wings, mushrooms on the cell walls and so on. Chitosan, has 2-amino-2 deoxy-d-glucose (glucosamine) monomers obtained through partial deasetilation of the chitin, connect with β-1,4 bonds, have reactive functional amino and hydroxyl groups, similar to cellulose as a chemical structure and most common biopolymer after cellulose in nature. Kitosan has bioactive properties such as hemostatics, bacteriostatic, fungicaatatic, anticanceorogen, wound and bone healing. Application of chitosan include films, hydrogels, fibers, micro/nanocapcules, and micro/nanoparticles. 

Currently, magnetic oxide nanoparticles attract great attention due to comprehensive practices ranging from basic research to industrial use. Spinel ferrite nanocrystals are considered to be two of the most important inorganic nanomalzema due to electronic, optics, electrical, magnetic and catalytic properties. Spinel Ferrites have the AB2O4 structure in which A and B show tetrahedral and octahedral cation regions, respectively and show the oxygen anion region. Metal spinel ferrite nanoparticles have general molecular formula MFe2O4 (such as Mg) and have a face -centered cubic structure. Since the magnetic separability plays an important role in expanding the medical applications, magnetic nanoparticles have been recently used in drug carrier systems. The main objective of this research was to develop chitosan-modified magnetic nanoparticles for drug delivery. ZnFe2O4, which is a paramagnetic and antibacterial nanometal oxide was used in this study. 
Imatinib is one of the drugs used in primary treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). Imatinib mesylate (commercial name Glivec) is a derivative of 2-phenilaminopyirimide. Imatinib Abl, C-Kit and PDGF-R (Platelet-derived growth factor receptors) have been developed as a specific tyrosine kinase kinase inhibitor. Tyrosine kinase activity is decreased by type I protein kinase inhibitors, which belong to this class. Tyrosine kinases are essential for cell development, proliferation, apoptosis, and the transmission of intracellular stimulation. They convert phosphorus from ATP to amino acids. As opposed to protein kinase inhibitors, imatinib binds to both the ATP binding zone and the hydrophobic region created in the comfort, causing the enzyme to "freeze" in the comformational inactive state.
Patients who use standard imatinib tablets have significant toxic/side effects as one of their greatest drawbacks. Due to the absence of patient cooperation and these adverse effects, severe side effects result in treatment cessation. Lower therapeutic concentrations of imatinib are caused by variation in its pharmacokinetics, which results in treatment failure and encourages drug resistance. There are studies that support the necessity for controlled release imatinib formulations in the literature. Imatinibin controlled release formulations were created as a result to enhance patient plasma levels.
The ionic gelation technique was implemented to prepare the nanoparticles. The prepared nanoparticles was loaded with imatinib, which is a tyrosine kinase inhibitor also known as "Glivec". The imatinib‐loaded ZnFe2O4-CS NPs were then used to investigate the drug encapsulation efficiency and in vitro drug release. 
The encapsulation efficiency was found to be 77.8%. The CS NPs prepared without ZnFe2O4 was also investigated for drug encapsulation. However, its efficiency was significantly lower (36%) than that of  ZnFe2O4 included CS NPs. 
For the characterization of the carrier, ZnFe2O4-CS NPs, FTIR, the particle size and transmission electron microscope analysis was performed. The FT-IR spectrum of imatinib has characteristic peaks at 3329.88 cm-1, indicating the presence of primary N-H in the construct, at 2966.23 cm-1, the presence of carboxylic acid C=O & O-H stretching in the construct. Chitosan shows a broad peak at 3359.87 cm-1, indicating the presence of a carboxylic acid group (R-COOH) within the chitosan structure. 
Chitosan alkanes show C-H stretch at 2966.11 cm-1 & 2876.13 cm-1. The strong intense peak at 959.55 cm-1 in chitosan IR spectra indicates the presence of ester (R-O-R) in its structure. The peak formation at 2936.34 cm-1 for IMT-PNPs indicates possible binding of the primary -NH group with the carboxylic chitosan group.
The physical mixture of imatinib and chitosan shows almost the same peaks indicates the absence of any drug-polymer interactions.
Scanning Electron Microscobe was used and it was observed that the average particle size of the nanoparticles containing chitosan coated ZnFe2O4 obtained as a result of SEM images was 23 nanometers. In this way, it has been proven that the composite material obtained by achieving the desired goal can be a drug carrier in nano size. The use of the drug carrier at the nanoparticle level in the encapsulation of the active substance is predicted to be the most efficient step for controlled drug delivery and release. In this way, the carrier loaded with nano-sized active substance will reach the diseased area more easily and effectively, and negative drug accumulations that may have side effects in the body will be prevented.
In the last stage, controled release of the composite material to the PBS (phosphate buffered saline) buffer which was simulated body environment was made with a dialysis membrane, and the amount of drugs in the samples taken from the buffer solution were determined and the release percentages were found. At this stage, the the success for but the controlled relase of the drug, not by a sudden release, was proven.
In this way, the optimum conditions required for controlled drug release developed with the combination of different techniques have been proven many times. The results obtained will shed light on the production of new formulations in the pharmaceutical industry and shed light on the studies to treat diseases with higher efficiency. This work could be helpful to the cancer researches which is on the overcoming the side effects of conventional chemotherapy.