Kanser teşhis ve tedavisinde kullanılma potansiyeli olan aza-BODIPY içerikli poli(β-amino ester)'lerin hazırlanması ve biyolojik özelliklerinin araştırılması
Preparation and evaluation of biological properties of poly(β-amino ester)s that have potential in cancer therapy and diagnosis
Yazar: ŞEYMA SARI
Danışman: DOÇ. DR. MUHAMMET ÜBEYDULLAH KAHVECİ
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Kimya Ana Bilim Dalı / Kimya Bilim Dalı
Derece: Doktora, 2024
Konu: Kimya = Chemistry
Dizin: Biyopolimerler = Biopolymers ; Fotokemoterapi = Photochemotherapy ; Poli beta amino ester = Poly beta amino ester

Özet:
Kanser, hem ölüm ve vaka sayılarının hem de teşhis/tedavi maliyetlerinin yüksek olması nedeniyle günümüzün sağlık sorunlarının başında gelmektedir. Geleneksel tedavi yöntemleri arasında önemli bir yere sahip olan kemoterapide kullanılan ilaçların sistemik toksisiteleri ciddi yan etkilere ve komplikasyonlara sebep olmaktadır. Buna ek olarak ilaçların çözünürlük problemleri, metabolize olması, ilaç direnci gelişmesi veya böbreklerden eliminasyonu gibi farmokokinetik parametreler çerçevesinde ilaç biyoyararlanımı ve etkinliği istenilen düzeylerde olmamaktadır. Bu sebeplerden ötürü kanserin teşhis ve tedavisine yönelik birçok çalışma bulunmaktadır. Polimerlerin birçok avantajlarından dolayı son yıllarda polimerlere dayalı tedavi ve teşhis  yaklaşımlarını da klinik uygulamalarda görmek mümkündür. Bu bilgiler ışığında ilaç ve ilaç salım sistemlerinde önemli bir sınıf olan polimerik malzemelere dayalı ilaç ve ilaç salım sistemlerinin geliştirilmesi oldukça önem arz etmektedir.  
Bu tez çalışmasında yapısında kızılötesine yakın (NIR) ışığı absorblayan/ışıma yapan aza-BODIPY molekülünü taşıyan, suda çözünür/dağılabilir, biyouyumlu, biyo-parçalanabilir ve pH'a duyarlı poli(β-amino ester)ler (PBAE) geliştirilmesi hedeflenmiştir. Bu amaç doğrultusunda uygun aza-BODIPY türevleri sentezlenerek Michael Katılma reaksiyonuna dayalı polikondenzasyon yoluyla aza-BODIPY içerikli bazı tür kanser hücrelerini hedefleyici özellik gösteren folik asit taşıyan ve yapısındaki aza-BODIPY ile floresans özellik sergileyen hidrofilik ve amfifilik karakterlere sahip PBAE'ler sentezlenmiştir. Sentezlenen küçük moleküller ile polimerlerin kimyasal ve fotokimyasal karakterizasyonları 1H/13C-NMR, FT-IR, UV-Vis ve floresans spektroskopileri yardımıyla gerçekleştirilmiştir. Polimerlerin molekül kütlesi ve morfolojik (misellerin) özellikleri GPC, DLS ve taramalı/geçirimli elektron mikroskobu (STEM) ile incelenmiştir. İlaç yükleme ve salım profilleri UV-Vis spektroskopisi özelliğine sahip mikroplaka okuyucu ile takip edilmiştir. Hidrofilik karakterli PBAE'lerin diyagnostik amaçlı hücre hedefleme/görüntüleme, PDT ve genetik materyal taşıma etkinlikleri  HeLa ve U87-MG kanser hücre hatlarında in vitro sitotoksik özellikleri araştırılmıştır. Hedefleme/görüntüleme çalışmaları floresans mikroskop ile takip edilmiştir. Amfifilik PBAE'lerin misel oluşturma, ilaç yükleme (doksorubisin) ve salım profilleri incelenerek misellerin kombine terapi (kemoterapi + PDT) ve teranostik platform potansiyeleri incelenmiştir. PDT, kombine terapi ve teranostik uygulamaları karanlık ortamda veya ışıkla aydınlatma sonrasında hücre canlılığı testleri yapılarak takip edilmiştir. Sentezlenen polimerik yapılar ilk kez tez kapsamında sentezlenmiş olup kanser teşhis ve/veya tedavisinde kullanılabilecek sistemlere katkı sağlama noktasında yüksek potansiyele sahip oldukları değerlendirilmiştir. Önerilen taşıyıcı platformun, gerek aktif hedefleme gerekse boyuta dayalı pasif hedefleme yaklaşımları ile kanser tedavisinde sıkça karşılaşılan yan etkilerin azaltılmasına olanak veren taşıyıcı sistemlere katkı sağlayacak potansiyele sahip olduğu görülmektedir. Ayrıca, geliştirilen polimerik yapıların PDT ve kombine terapi yaklaşımları, kemoterapide rastlanan ilaç direncine çözüm önerisi getirme potansiyeline sahip olduğu görülmüştür. Bu tez çalışmasında model hedefleyici ligant olarak folik asit ve model hücre hatları olarak HeLa ve U87-MG seçilmiştir fakat farklı ligantlar ile farklı kanser hücrelerinin hedeflenebilir olması nedeniyle bu sistemler, diğer çalışmalar için modüler bir platformun temelini oluşturmaktadır.

Abstract:
Although there have been many chemical and technological developments today, there are still many diseases waiting to be treated. Cancer, which has the most important place among these diseases, is one of today's health problems due to the high number of deaths and cases as well as diagnosis/treatment costs. It is seen that millions of people die due to cancer every year around the world. The most important problem during the treatment of cancer is that the chemotherapeutic drugs/substances used have an integrated effect instead of a local effect. Indeed, chemotherapeutic drugs/substances that have toxic effects to destroy cancerous cells, while destroying cancerous cells, also destroy healthy cells. In addition to killing healthy cells, this situation also limits the drug dosage that can reach the tumor area. As the drug reaching the cancerous area decreases, the patient is exposed to more (number of doses) or higher doses of chemotherapeutic drugs/substances. On the other hand, due to this toxic level, serious side effects are observed in patients in addition to weakening of immunity. Due to these side effects, the patient may have to discontinue the treatment either compulsorily or optionally. Chemotherapy, which has an important place among traditional cancer treatment methods, can cause many side effects such as fever, nausea, vomiting, fatigue, diarrhea, pain, hair loss, anorexia nervosa, anxiety, sleep disorder, depression and other conditions that affect the quality of life of patients. In the light of this information, since the development of a new drug molecule is a high-cost, time-consuming and laborious process, the development of targeted drug delivery systems that can increase the dosage in the cancerous area by reducing the side effects of 'old' drugs with side effects; It will significantly shorten the process of applying personalized treatment methods to the patient and allow many more patients to benefit from the treatment systems. On the other hand, the development of treatment methods that allow local treatment will further increase this effect. Non-invasive, light-triggered phototherapy, developed in recent years, is used in clinical studies for cancer and some other diseases. Phototherapy includes photodynamic therapy (PDT) and photothermal therapy (PTT). PDT is based on the attack of reactive oxygen species (ROS), which are formed as a result of the stimulation of a photo-stimulating molecule with light (600-800 nm), on biological molecules and ultimately the prevention (inhibition) of the proliferation of target (cancer) cells, and the ROS formed in the light environment cause apoptosis. It destroys tumor cells by causing necrosis or autophagy. One of the most important advantages of PDT is that it provides temporal and spatial control during treatment, reducing side effects and complications, and offering a more comfortable treatment opportunity to the patient. On the other hand, it is also very important to develop systems that allow combined treatment with PDT. It is also of great importance to develop systems suitable for combined therapy, where chemotherapy and PDT are used together, in order to reduce side effects and develop a more effective treatment system. 
Polymers are one step ahead of other materials in many applications due to their advantages. Fluorescence polymers, an important class of polymers, are defined as polymers containing fluorophore groups in their structure. Thanks to their fluorescence properties, such polymers are frequently used in chemical identification and tracking processes. As in many fields, fluorescent materials are needed for chemical identification and tracking in the field of healthcare. There are some properties that polymers must have in order to be used in applications in biological environments. Except for exceptional cases, fluorescent polymers are required to be water-soluble/well-dispersible, biocompatible and generally biodegradable. In summary, there is an increasing interest in the design and synthesis of biodegradable and water-dispersible/soluble fluorescent polymers in biomedical fields such as drug delivery, imaging, diagnosis, and tissue engineering. Polymeric carriers also play a role in drug release that can be triggered by environmental effects (pH, temperature, light, etc.). Providing controlled release in this way also functions to reduce side effects. The fact that the pH in cancer tissue or cells (<6.5) is lower than physiological pH (7.4) has led to the development of pH-sensitive polymeric carriers. On the other hand, biodegradable polymers constitute an important polymer class that attracts much attention. Due to their biodegradability, they are at the center of biomaterial science for many biomedical applications such as drug delivery, imaging and diagnosis. In this area, biodegradable polymers are gaining importance. The most studied type of biodegradable polymers, of which there are many types such as polyanhydrides, polyurethanes, poly(ester amide)s, are polyesters. Polylactides, polyglycolides and their copolymers are frequently used polyesters approved by the American Food and Drug Administration (FDA). In recent years, poly(β-amino esters) (PBAEs) have become very interesting for biomedical applications such as carrying genetic material or therapeutic agents due to their non-toxicity and functionalizability. On the other hand, they are good candidate carriers for the triggerable release of anti-cancer therapeutics due to their pKb values around 6.5. PBAEs have recently attracted great attention due to their natural biocompatibility and biodegradability, and PBAEs and acrylates are obtained by amine addition and Michael addition in a single step, without the formation of any byproducts. They have polymer properties suitable for various biomedical applications such as anticancer drugs, antimicrobials, protein delivery and tissue engineering. Compared to classical fluorescent dyes, boron complexes of dipyromethene (BODIPY) and azadipyromethene (aza-BODIPY) are dyestuffs with strong absorption in the visible region, narrow emission band with high quantum yield, and good physiological and photochemical stability. aza-BODIPYs have attracted more attention than their analogues due to their superior photochemical properties, as light in the near-infrared region can penetrate biological environments to a certain depth. This shows that such materials can be excited or a signal can be received from the dye molecule even within the tissue, and in order to be used in the biological environment, such fluorescence dyes must be water-dispersible or soluble. Due to the nature of the tumor tissue, the gap between the endothelial cells in the blood vessel wall is larger than in normal tissues, which increases the vascular permeability in the tumor area and increases the passage of relatively large molecules (for example, polymeric nanoparticles) into the tumor area. Again, the absence of a lymphatic system and therefore lymphatic drainage in the nature of the tumor tissue increases the accumulation of substances in this region. As a result of these two features, it is possible to accumulate therapeutic agents in the tumor area with the help of appropriately sized carriers with the "increased permeability and retention (EPR) effect". It is aimed that the polymeric structures to be developed within the scope of the thesis will accumulate in the tumor area and provide passive targeting with a similar approach. Thus, side effects of therapeutic agents can be reduced due to the principle of release to the tumor site. On the other hand, it is planned to design and synthesize PBAE-based biodegradable aza-BODIPY-containing polymers that can "actively target" cancer cells. Folic acid is a ligand frequently used in identifying or targeting cancer cells. Many malignant tumors express excessive amounts of folate receptors on their cell surface. As a result, the side effects of therapeutic agents can be reduced more effectively by using a carrier system that can selectively passively and actively target tumor tissue, thanks to a carrier containing targeting ligands prepared in an appropriate size.
Therapeutic agent delivery systems integrated with fluorescence imaging, which are the basis of reliable approaches that play a role in making the biological environment visible in vitro or in vivo, are prominent platforms for tumor diagnosis and therapy. In other words, theranostics can be defined as systems that simultaneously carry the therapeutic agent and the diagnostic imaging agent in the same dose. In such systems, therapy can be provided with medication (chemotherapy) and genetic material (gene therapy), as well as alternatives such as phototherapy, radiotherapy or thermal therapy. Instead of using separate materials for treatment and imaging, theranostics that carry both functionalities on the same platform can reduce possible side effects. The most important purpose of the use of theranostics is to monitor the condition of the diseased area with imaging ability, to monitor the release kinetics and drug effectiveness, and to adjust the appropriate therapy and dose. This makes a significant contribution to the development of a personalized medicine approach with appropriate doses, as opposed to applying the same single dose to all patients.
In this thesis, it is aimed to develop water-soluble/dispersible, biocompatible, biodegradable and pH-sensitive poly(β-amino esters) (PBAE) carrying aza-BODIPY molecule which absorbs/emits near-infrared (NIR) light in their structure and to investigate their potentials for tumor cell targeting/imaging, gene therapy, phototherapy, combined therapy and theranostic applications. The polymeric structures designed very first time in this thesis are considered to have a high potential to aid cancer diagnosis and/or treatment systems. It is envisioned that the proposed carrier platform will contribute to carrier systems that enable the reduction of side effects commonly encountered in cancer treatment with both active targeting and size-based passive targeting approaches. Moreover, the photodynamic therapy (PDT) and combined therapy approaches proposed in this thesis have the potential to address drug resistance, another common issue encountered in chemotherapy. In this thesis, folic acid was selected as the model targeting ligand and HeLa and U87MG as the model cell lines. However, since different cancer cells can be targeted with different ligands, the system developed within the scope of the thesis forms the basis of a modular platform for further studies. Both hydrophilic and amphiphilic PBAEs carrying folic acid in their end groups were designed. aza-BODIPY-containing PBAEs were prepared by polycondensation polymerization via Michael addition reaction. Small molecules and polymers were characterized by 1H-13CNMR, FT-IR, UV-Vis and fluorescence spectroscopies. Molecular mass and morphological (micellar) properties of the polymers were investigated by GPC, DLS, scanning/transmission electron microscopy (STEM). Drug loading and release profiles were monitored using a microplate reader equipped with UV-Vis spectroscopy capability. The cell targeting/imaging, PDT and genetic material transport activities of hydrophilic PBAEs were investigated. The in vitro cytotoxic properties of these PBAEs were examined in HeLa and U87-MG cancer cell lines. The targeting/visualization studies were monitored with a fluorescence microscope. Micelle formation, drug loading (doxorubicin) and release profiles of amphiphilic PBAEs were investigated and the potential of micelles for combined therapy (chemotherapy + PDT) and theranostic platform were examined. PDT, combined therapy and theranostic applications were followed by cell viability tests conducted in the dark or after illumination with light.