Hedefli kanser tedavisine yönelik kil katkılı nanopartikül üretimi ve modellenmesi
Production and modeling of clay additive nanoparticle for targeted cancer treatment
Yazar: DENİZ KARATAŞ
Danışman: PROF. DR. MEHMET SABRİ ÇELİK ; DOÇ. DR. ADEM TEKİN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Cevher Hazırlama Mühendisliği Ana Bilim Dalı / Cevher Hazırlama Bilim Dalı
Derece: Doktora, 2017
Konu: Biyokimya = Biochemistry ; Biyomühendislik = Bioengineering ; Biyoteknoloji = Biotechnology

Özet:
İnsanın çevresel ve biyolojik etmenler nedeni ile yaşamı boyunca sağlığı bozulmakta, çeşitli hastalıklara maruz kalmaktadır. Günümüzde insanları en çok tedirgin eden ve tıp/ farmakoloji bilimi çevrelerince de en çok araştırma konusunu oluşturan, kanser hastalığı ve türlerinin tedavi yöntemleri çalışmalarıdır. İnsanlar bu hastalıkların tedavisi için doktor tavsiyesinde ilaçlı/ilaçsız farklı terapilere başvurmaktadır. Çoğunluğu ilaç ile olan bu tedavilerde, ilacın yan etkileri ve hasta-uyumlu olması çok önem arz etmektedir. Bilim insanları da sürekli, hem deneysel hem klinik olarak daha etkili; doğru ilacı, uygulama dozajını ve şeklini bulmanın araştırmalarını sürdürmektedir. Ancak, kanser kemoterapisinde kullanılan ilaçların hem kimyasal yapıları hem de uygulama sıklığı hastada kemik erimesi, saç dökülmesi gibi yan etkilere sebebiyet vermekte ve iyileşme süresini de uzatabilmektedir. 
Son yıllarda ilaç taşıma sistemleri konulu birçok çalışma gerçekleştirilmiştir. Yapılan bu araştırmalarda temel amaç, tedavide kullanılan etken maddelerin daha az yan etkili ve biyo uyumlu kompozitlerle hastaya verilmesi ve tedavi süresinin kısaltılması yönünde olmaktadır. Bu ilaç taşıma sistemleri ağız yoluyla (oral) gerçekleştirildiği gibi özellikle kanser tedavileri için uygun olan damar yolu (i.v.) ile de yapılabilmektedir.
Günümüzde yetkili merciler tarafından klinikte kullanımı onaylanan nano-ilaç taşıma sistemlerinin çoğu amfifilik malzemelerden oluşan ve kendiliğinden birleşme (Self Assembly; SA) özelliğine sahip olan küreciklerden ibarettir. Bu amfifilik malzemeler arasında Poli laktik ko-glikolik asit (PLGA) günümüzde iyi profili ile en bilinen polimerik yapılardan biridir. PLGA ve benzeri malzemelerden oluşan ilaç taşıma sistemleri, birçok avantaja sahip iken, "kendiliğinden birleşme" davranışının bir dezavantajı olarak, hızlı kan akışında dağılarak, taşıdıkları ilacı tümör bölgesine varmadan serbest bırakırlar. Bu sorunu en aza indirgemek ve daha kararlı bir formülasyon geliştirmek için doğal kil mineralinin tabakalı yapısından birleştirici klips gibi yararlanılabilir. Sodyum-Montmorillonit (Na-MMT) biyo-uyumlu, kolay elde edilebilir ve amfifilik materyallerle etkileşebilen tabakalı bir yapıdır. Hedefli kanser tedavisi için nanopartikül boyutunun 200 nm civarında olması gerektiği açıklanmıştır. Ancak bugüne kadar yapılmış olan çalışmalarda genelde 300 nm civarında partiküller üretilmiştir. Dolayısıyla bu ihtiyacı karşılamak için bu çalışma gerçekleştirilmiştir.
Pasif hedeflendirme, ilgili dokunun fizyolojik özellikleri dikkate alınarak gerçekleştirilmektedir. Hastalıklı olan bölgedeki kan damarlarının çeperini oluşturan endotelyal hücrelerinin arasındaki mesafenin normalinden daha büyük olması nanotaşıyıcının pasif olarak bu bölgeye sızmasını ve birikmesini sağlamaktadır. Böylece etkin madde gelişmiş geçirgenlik ve alıkonma etkisi (EPR) sayesinde pasif hedeflendirilmiştir.  Bu çalışmadaki hedefimiz de kil katkılı olarak 200 nm altında üretilecek nanopartikülün doğal akışı içerisinde pasif olarak kanserli bölgeye hedeflendirmekti.
 
Bu hedef doğrultusunda bu tez çalışması deneysel ve teorik olarak iki aşamada gerçekleştirilmiştir. Deneysel çalışmalar öncelikle 122nm boyuta sahip olan PLGA/Na-MMT kompoziti elde edilerek başlanmıştır. Daha sonra bu nanokompozitin ilaç taşıma davranışını incelemek üzere, anti-kanser özelliği ve spektrofotometrik ölçümlerdeki kolay kullanımı ile bilinen, Curcumin, bu kompozitlere yüklenmiştir. Bu kapsamda boş kürecik elde etmek için öncelikle Na-MMT' in boyutu mekano-fiziksel yollarla nanometre ölçeğine indirgenmiştir.  İlaç taşıyan partiküllerin üretiminde ise önce Curcumin PLGA' ya aseton ortamında yüklenip daha sonra saf su ortamında hazırlanmış olan kil ile Diyaliz Membranı (DM) kullanılarak misel formunda sağlam yapılı bir nanokompozit elde edilmiştir. Yapılan karakterizasyon çalışmaları sonucunda bu nanokompozitin 1ml'de 1250 μg Curcumin taşıma kapasitesi olduğu, bu miktardan 812 μg'ın efektif olarak nano taşıyıcının içinde olduğu sonuçları elde edilmiştir. Bu formülasyonun MCF-7 hücre soyu üzerinde gösterdiği IC50 değeri 100μg olarak tespit edilmiş ve boş nano taşıyıcının L-929 fibroblast hücreleri üzerinde dikkat çekici toksik etkisi gözlenmemiştir. Benzer çalışmalar 5-Flourourasil ilacı ile de tekrarlanmış ve bu ilacın da hazırlanan kompozit ile taşınabildiği ve toksik etkisinin olmadığı ortaya konmuştur. 
Tez kapsamındaki teorik çalışmaların ana hedefi ise deneysel olarak üretilmiş olan bu kompozitin bileşenlerinin atomik ölçekte biribiri ile olan ilişkisini ortaya koymaktı. İlk olarak kuantum mekanik hesaplamalar ile PLGA polimerinin yapı taşları arasındaki dizilimin ne şeklinde olduğu açıklanmaya çalışılmış daha sonra sırasıyla Curcumin ve MMT ile olan moleküler arası etkileşimleri hesaplanmıştır. Bu hesaplar neticesinde elde edilen bağlanma enerjileri her üç bileşenin de birbiri ile etkileştiğini ortaya konmuştur. Ancak daha kesin ve doğru sonuç vermesine karşın deneysel kurguya daha çok yakınlaşmak ve etkin mekanizmayı açıklamak amacıyla Moleküler Dinamik (MD) simülasyonları da gerçekleştirilmiştir. Curcumin ile yapılmış kuantum hesaplamaları ve moleküler dinamik benzetimleri birlikte değerlendirildiğinde, kilin Curcuminle daha güçlü bir etkileşim halinde olduğunu ve PLGA' nın ise Curcuminle yakın temasta olan kil tabakasını sarmalamaya çalıştığı sonucuna varılmaktadır. Aynı zamanda, Curcumin moleküllerinin tümünün kil yüzeyine yerleşmediği bir kısmının PLGA sarmalı içerisinde de yer aldığı da gözlemlenmiştir. Benzer sonuçlar 5-FU ilacı için de gözlenmiştir. Bu teorik çalışmalarda H-bağları, van der Waals ve Coulomb etkileşimleri gözlenmiş ancak en etkini elektrostatik etkileşimler olduğu saptanmıştır.
Sonuç olarak, tez çalışması boyunca yapmış olduğumuz deneysel çalışmalarımızda ilk olarak tamamen fiziksel yollarla kil minerali nano boyuta indirgenmiştir. Bu partiküller vasıtasıyla hazırlanan nano kompozitin hedefli kanser tedavisinde kullanılabilecek formda olduğu ortaya çıkarılmıştır. Öte yandan yapılmış olan atomistik modellerden de kilin ilaç ve PLGA için uygun bir yüzey olduğu ve ilaç taşımanın yanında salımına da katkı sağladığı belirlenmiştir.

Abstract:
Human health is the most important value we have. Starting from the individual, any kind of material and psychological problems that will occur in the society will also affect the backwardness of the nations. The development and promotion of societies and nations is through having a collection of healthy individuals knowing this value.
Throughout life, the health of people is deteriorated due to the reasons of environmental and biological factors and they are exposed to various diseases. Today, the most disconcerting people and medicine / pharmacology is the study of the treatment methods of cancer. People refer to different therapies with medication on the recommendation of a doctor for the treatment of these diseases. In these treatments with the majority of medications, side effects of the medication and patient compliance are very important. The details related to the investigations are also more effective, both experimentally and clinically, such as the right medicine, the dosing of the application and the shape of the drug. However, both the chemistry and the application frequency of drugs used in cancer chemotherapy cause side effects such as bone erosion and hair loss in the patient and can prolong the healing period. 
In recent years, many studies have been carried out on drug delivery systems. In these investigations, the main idea is to give the active ingredients with biocompatible composites used in the treatment with fewer side effects and to shorten the duration of the treatment. These drug delivery systems can be made by the oral route or intravenous (i.v.), which is especially suitable for cancer treatments. 
In this dissertation, an experimental and theoretical study was carried out on the targeted drug release of curcumin and 5-Fluorouracil (5-FU) drugs used in cancer chemotherapy using modified montmorillonite cages with ore preparation methods. Curcumin is a fluorophore compound derived from rhizomes of Curcuma longa (Turmeric) plant, known for its antioxidant, anti-Alzheimer, anti-cancer and similar biological activities. In many studies up to date, compounds with high fluorescence emissions such as pyrene, curcumin have been used to study the drug transport capacity of nano-drug delivery systems, followed by other studies with cytotoxic effects to investigate in-vitro anti-cancer. The substance such as paclitaxel and 5-FU has been loaded into these systems. In this study, Curcumin and 5-FU were used at all stages to make all the physico-chemical and biological activities of the PLGA-clay nanocomposite we created as a new product and to illuminate the unexplained points in detail with quantum chemical calculations and Molecular Dynamics (MD) studies. 
5-FU undergoes liver when used orally. When used intravenously, it is distributed throughout the body and the brain blood barrier, as well. 5-FU circulating throughout our body in about 1 hour and thus some side effects are caused such as hair loss, liver  
failure, anemia and bone marrow depression. A clay and polymer-based nanoparticle has been produced throughout our work to reduce these side effects.
As is known, materials, physical chemistry and surface chemistry are the main disciplines that Mineral Processing Engineering works together. Today, clay-based hybrid materials such as sepiolite, kaolin and montmorillonite are produced using mineral preparation and chemical techniques. These materials are frequently used in advanced technological fields. One of the most important of these uses is the health sector. The purpose of use is to reduce the side effects that may occur in classical drug treatments, by using the drug in lesser amounts and in the correct formulation. Because of the transport and release of medicines, in recent years, different disciplines have been working intensively jointly. Drug release in the human body can also be done by irradiation, such as via oral, nasal or skin routes. However, in the transport and release of drugs, it is important that the side effects of the carrier material are reduced and that the drug delivery to the targeted cell is more active and effective, and drug release is controllable.
Rapid developments in nanotechnology have shown that nanoparticles used in cancer treatment can also be formulated with inorganic materials other than organic materials. Clay minerals such as atapulgite, double-layered hydroxides (LDH) and halloysite are widely used inorganic materials. These clay minerals, which are generally aluminum-magnesium hydrosilicate (Al, Mg-SiO2.nH2O), are being used extensively in medical and pharmacological applications due to their layered structure, large surface area and high amount of variable cation capacity. Prior art has been used for oral or topical applications in the preparation of formulations such as capsules, tablets, suspensions, emulsions using rheological and sorptive properties of the clay minerals. In recent years, crystal of kaolinite, sepiolite and montmorillonite has been used as a direct or auxiliary material in the formation of nanoparticles. Natural, hydrophilic and non-toxic inorganic minerals are also used for drug release.
By means of a cation exchange mechanism, a cationic drug was loaded onto a negatively charged surface clay and released in vivo. However, in order to realize this loading at the nano size, the clay must be able to be reduced to the nano size. Therefore, there is a need for a clay that can be easily layered by physical methods such as decantation, mechanical mixing and centrifugation. This clay can be montmorillonite (MMT) mineral which has great reserves in Tokat province in Reşadiye region in Turkey (commercial name is bentonite). Clay will play a reinforcing role in the nanoparticle to be prepared and is thought to help create a global grain. The MMT clay is soft, plastic, porous and light colored. MMT, which has a very large surface area, can be characterized by acid activation. Due to its properties such as colloidal properties when mixed with water, its swelling in water, its high plasticity and its ion exchange capacity, it can be used for drilling mud elation, bleaching of edible oils, water-solvent rheological additive, waste water cleaning, animal feed additives, nuclear waste disposal, nanocomposite production.
In pharmacology, drug transport and release experiments are carried out mainly with polymer in the in-vitro and in-vivo (living cell) environments. However, the side effects of these dosage systems, their chemical content and their high costs, encourage the search for alternative carrier systems. In this study, a nanoparticle formulation was developed for use in targeted cancer treatment using Poly lactic co-glycolic acid (PLGA) polymer, sodium montmorillonite (Na-MMT), a natural raw clay mineral, curcumin and 5-FU drugs and this nanoparticle theoretically modeled with the density function theory (DFT) and by Molecular Dynamics (MD) simulation techniques.
Most of the nano-drug delivery systems currently approved for use in clinical practice by competent authorities are spheres composed of amphiphilic materials due to the self-assembly (SA) properties. Among these amphiphilic materials, Poly lactic co-glycolic acid (PLGA) is one of the most famous polymeric structures known today with good profile. However, drug delivery systems composed of PLGA and similar materials have many advantages, but due to the disadvantage of "self-assembly" behavior, they are dispersed in the rapid blood flow and released without reaching the tumor site of the drug they are carrying. To minimize this problem and develop a more stable formulation, the stratified structure of the natural clay mineral can be utilized like a joining clip. Sodium-Montmorillonite (Na-MMT) is a biocompatible, easily obtainable, layered structure that can interact with amphiphilic materials. It has been demonstrated that the nanoparticle size should be around 200 nm for targeted cancer treatment. However, in the studies carried out so far, particles of around 300 nm were produced in general. Therefore, this work has been done to solve this main problem. 
As it is well-known, passive targeting is performed by taking into account the physiological characteristics of the respective tissue. The fact that the distance between the endothelial cells forming the wall of the blood vessels in the diseased area is greater than the normal, allows the nano drug carrier to passively infiltrate and accumulate in this region. Thus, the active substance is passively targeted by enhanced permeability and retention effect (EPR). Our aim in this study was to passively target the cancerous region in the natural flow of the caly additive nanoparticle to be produced under 200 nm.
This study was conducted experimentally and theoretically in two stages. Experimental studies were initiated by obtaining a PLGA / Na-MMT composite having a size of 122 nm. Curcumin, which is known for its easy-to-use anti-cancer properties and spectrophotometric measurements to examine the drug transport behavior of this nanocomposite, is then loaded into these composites. In this context, the size of Na-MMT was first reduced to nanometer size by mechano-physical means to obtain empty spheroids. In the production of drug-bearing particles, a nano-composite was first obtained in Curcumin/PLGA in acetic acid and then in a micelle form using a dialysis membrane (DM) with clay prepared in pure water. As a result of the characterization studies carried out, it was found that this nanocomposite had 1250 μg Curcumin transport capacity in 1 ml, 812 μg from this amount was effectively contained in the nanocarrier. The IC50 value of this formulation on the MCF-7 cell line was determined to be 100 μg, and no remarkable toxic effect was observed on the L-929 fibroblast cells of the empty nano-carrier. Similar studies have also been repeated with 5-Fluorouracil and it has been demonstrated that this drug can be transported with the prepared composite and carries no toxic effect. The IC50 value of the PLGA-Clay-5-FU nanocomposite was calculated as 21,30 μg/ml. This value has been found in the literature to be approximately twice that of the chitosan-MMT nanoparticle containing 5-FU compared to the A549 cell line (11,49 μg / ml) and it has been found that 5-FU molecules can be loaded more with the particle we prepared.
As a result of experimental studies, this nano drug carrier produced by this method for the first time in the world and able to be used in cancer treatment. It is a combination with polymeric material without the need for clay exfoliation. It is produced as nanocomposite acquisition without centrifugation. Nanoparticle obtains higher drug loading capacity and less toxic nanocomposite formation and it is produced for nano drug delivery system with ideal release behavior for targeted cancer treatment.
In all the experimental studies carried out in summary, the relationship between clay, PLGA and drug trio has been revealed on a macro scale, but interaction mechanisms and formation at the atomic level are a big question. Therefore, this nanoparticle has been investigated theoretically in this study.
The main objective of the theoretical studies in the thesis is to reveal the relation of these experimentally produced composite components to each other at atomic scale. Initially, quantum mechanical calculations revealed that interaction between chains related to PLGA polymer was observed, and then interactions between molecules with Curcumin and MMT were calculated, respectively. The binding energies obtained from these calculations show that all three components are compatible with each other and interact. However, Molecular Dynamics (MD) simulations have been carried out in order to explain interactions, although quantum mechanics studies are more accurate. When we evaluate the quantum calculations and molecular dynamics simulations made with Curcumin together, we conclude that MMT is in stronger interaction with curcumin and that PLGA is trying to wrap some clay layer, which is close to curcumin. It has also been observed that a portion of the curcumin molecules, not all of which are deposited on the clay surface, are also located within the PLGA cohesive. Similar results were observed for the 5-FU drug. 
In addition to the experimental work, the theoretical studies revealed the interaction between the particle components produced. In the quantum chemical models, it was seen that the PLGA chain formed coil. In these expanded models, it has been found that the drug / PLGA / clay triple is associated with electrostatic interactions. When models are observed with dynamic simulations at the molecular level, it has been shown that drug molecules can be attached to both clay and PLGA spins, and all these model analyzes reveal that clay can be a suitable surface and drug carrier mineral for this particle. In brief, in these theoretical studies, H-bonds as well as van der Waals and Coulomb interactions were observed, but the most interaction was electrostatic interactions.
As a result, in our experimental studies that we have conducted throughout our thesis work, the clay mineral was first reduced to nano size in pure physical ways. It has been discovered that the nanocomposite prepared by these particles is in a form that can be used for targeted cancer treatment. On the other hand, it has been stated that the atomic models built are suitable for deliviring and releasing drug molecules from the PLGA/MMT composites and contributing to a better understanding of experimental studies.