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Fotoakustik görüntüleme uygulamaları için altın ve BODIPY içeren lipozomal kontrast ajanlarının geliştirilmesi ve karakterizasyonu

Development and characterization of gold and BODIPY loaded liposomal contrast agents for photoacoustic imaging applications

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  1. Tez No: 1019709
  2. Yazar: ELİF GÜRBUZ
  3. Danışmanlar: PROF. DR. ÖZGÜR ÖZDEMİR, DOÇ. DR. ASİYE YURTTAŞ
  4. Tez Türü: Yüksek Lisans
  5. Konular: Biyomühendislik, Elektrik ve Elektronik Mühendisliği, Bioengineering, Electrical and Electronics Engineering
  6. Anahtar Kelimeler: BODIPY bileşikleri, Fotoakustik, Lipozomlar, BODIPY compounds, Photoacustic, Liposomes
  7. Yıl: 2026
  8. Dil: Türkçe
  9. Üniversite: İstanbul Teknik Üniversitesi
  10. Enstitü: Lisansüstü Eğitim Enstitüsü
  11. Ana Bilim Dalı: Elektronik ve Haberleşme Mühendisliği Ana Bilim Dalı
  12. Bilim Dalı: Biyomedikal Mühendisliği Bilim Dalı
  13. Sayfa Sayısı: Belirtilmemiş.

Özet

Günümüzde kanser teşhisinin ve tedavisinin eş zamanlı olarak yürütüldüğü“teranostik”(terapi ve tanı) çalışmaları onkolojik araştırmaların merkezinde yer almaya başlamıştır. Fotoakustik görüntüleme sistemleri teşhiste kullanılan optik kontrastın moleküler özgüllüğünden ve ultrasonun derin doku penetrasyonu özelliklerinden faydalanarak yüksek çözünürlüklü görüntüleme avantajıyla tümörlerin erken evrede tespitini sağlayabilir ve noninvaziv olarak tedavi takibine katkı sunabilir. Fotoakustik görüntüleme (FAG) belirli bir dalga boyundaki lazer emilimiyle oluşan termoelastik genleşmenin ultrason probuyla ölçülmesi prensibine dayanır. Bu görüntüleme sisteminin klinik başarısı, biyolojik sıvılarda bozulmadan hedef bölgeye ulaşabilen ve o bölgede teşhis için gerekli sürede güçlü sinyal verebilen kararlı yapıdaki kontrast ajanlarının ve uygun taşıyıcı sistemlerinin geliştirilmesine bağlıdır. Bu çalışmada kontrast ajanı olarak, 532 nm dalga boyunda yüksek optik emilim performansı gösteren, Yüzey Plazmon Rezonansı (SPR) özelliğine sahip altın nanoparçacıklar (AuNP) ile sunduğu fiziksel ve biyolojik avantajlar nedeniyle BODIPY organik boyası tercih edilmiştir. Bu nedenle her iki ajan da fotoakustik görüntüleme için son derece uygun kontrast maddeleri olarak değerlendirilmektedir. 532 nm uyarımı, her iki ajanın fotoakustik verimliliğinin maksimize edildiği ortak spektral noktada buluşarak tanısal hassasiyeti artırmaktadır. Tek başlarına agregasyon eğilimi gösteren bu ajanlar, biyouyumluluk ve uzun süreli stabilizasyonu sağlamak amacıyla lipozomal veziküllere hapsedilmiştir. Kontrast ajanları için taşıyıcı görevini üstlenen lipozomların formülasyonunda kullanılan soya lesitini, yapısındaki doymamış yağ asitleri sayesinde oda sıcaklığında akışkan bir zar yapısı sunarken, fiziksel stabiliteyi artırmak için literatürde sıklıkla yapıya kolesterol eklenmektedir. Eklenen kolesterol miktarı zar rijitliğini artırırken, buna bağlı olarak da termoelastik genleşmenin fiziksel kısıtlanması ile fotoakustik sinyal sönümlemesine yol açıp açmadığı literatürde yeterince incelenmemiştir. Çalışmamızda farklı kimyasal yapıya sahip ajanların kapsüllenme stabiliteleri ve kolesterolün fotoakustik sinyal üzerindeki etkisi karşılaştırmalı olarak incelenmiştir. Bu amaç doğrultusunda değişen molar oranlarda kolesterol içeren lipozomlar etanol enjeksiyonu yöntemiyle sentezlenerek AuNP ve BODIPY molekülleri kapsüllenmiştir. Sentezlenen lipozomların 1., 7. ve 14. günlerdeki periyodik fotoakustik ölçümlerinin zamana bağlı sinyalleri takip edilmiştir. Bu bağlamda kolesterol miktarının termoelastik genleşme üzerindeki kısıtlayıcı etkisi incelenerek sızıntı direnci ile sinyal genliği arasındaki ideal dengenin (%25 kolesterol oranında) bulunması ve daha yüksek oranların (%35) akustik sönümlemeye neden olduğunun ortaya konması amaçlanmıştır.

Özet (Çeviri)

Today,“theranostic”(therapy and diagnostics) studies, in which cancer diagnosis and treatment are conducted simultaneously, have taken center stage in oncological research. Photoacoustic imaging systems can facilitate the early-stage detection of tumors with the advantage of high-resolution imaging by utilizing the molecular specificity of the optical contrast used in diagnosis and the deep tissue penetration capabilities of ultrasound, thereby contributing to non-invasive treatment monitoring. Photoacoustic imaging combines the high molecular contrast of optical imaging with the deep tissue penetration of ultrasound. Photoacoustic imaging (PAI) is based on the principle of measuring the thermoelastic expansion caused by laser absorption at a specific wavelength using an ultrasound probe. The clinical success of this imaging system depends on the development of stable contrast agents and appropriate delivery systems that can reach the target area without degrading in biological fluids and generate a strong signal for the duration required for diagnosis in that area. Due to the high rate of light scattering, which is one of the biggest disadvantages of conventional light-based imaging methods, imaging in biological tissues generally remains superficial. Unlike conventional optical methods that can only penetrate to a depth of 1 mm, the photoacoustic system, which can penetrate up to 70 mm, offers detailed imaging from the organelle to the organ level with sub-micrometer resolution. Especially in areas where deep tissues such as the tumor microenvironment and angiogenesis need to be imaged, and where endogenous agents (naturally occurring agents in the body like hemoglobin, melanin, and lipids) are insufficient, high-contrast images can be generated using externally administered, specially designed exogenous agents. In this study, gold nanoparticles (AuNPs), which exhibit high optical absorption performance at a 532 nm wavelength and possess Surface Plasmon Resonance (SPR) properties, along with BODIPY organic dye, due to its physical and biological advantages, were chosen as exogenous contrast agents. Therefore, both agents are considered highly suitable contrast materials for photoacoustic imaging. The 532 nm excitation increases diagnostic sensitivity by converging at a common spectral point where the photoacoustic efficiency of both agents is maximized. Gold nanoparticles have the ability to generate a strong photoacoustic signal amplitude due to the collective oscillation of free electrons on their surface when exposed to laser light excitation. The BODIPY dye, on the other hand, is a frequently used contrast agent in the literature due to its high molar extinction coefficient. The small-molecule fluorophore structure of the agent offers high photochemical stability in biological environments. A common characteristic of both agents is that when administered into the bloodstream in a free form, they can be rapidly cleared from the body by the reticuloendothelial system (RES) and may exhibit a tendency to aggregate on their own; therefore, it is imperative to encapsulate the agents within liposomal vesicles to ensure biocompatibility and long-term stabilization. While soy lecithin, used in the formulation of liposomes serving as carriers for contrast agents, provides a fluid membrane structure at room temperature due to its unsaturated fatty acids, the addition of cholesterol to the structure is frequently recommended in the literature to increase physical stability. Cholesterol molecules added to the membrane intercalate between the acyl chains of the phospholipids, ensuring a more tightly packed membrane structure. As the packing density increases, the elastic modulus of the membrane against mechanical stress also changes. Although this structural tightening in the membrane plays an important role in preventing the premature leakage of encapsulated agents, it narrows the elastic space required for the volumetric expansion of the agents caused by laser light excitation. This rigidity in the membrane carries the potential to cause a mechanical damping effect by preventing the generated ultrasonic waves from propagating outward. While the added amount of cholesterol increases membrane rigidity, whether it leads to photoacoustic signal damping due to the physical restriction of thermoelastic expansion has not been sufficiently investigated in the literature. In our study, the encapsulation stabilities of contrast agents with different chemical structures and the effect of cholesterol on the photoacoustic signal were comparatively investigated. For this purpose, liposomes containing different molar ratios of cholesterol were synthesized using the ethanol injection method, and AuNP and BODIPY molecules were encapsulated within the liposomal membrane. Various techniques exist in the literature for the synthesis of liposomes, among which the thin-film hydration method, reverse-phase evaporation method, and ethanol injection method are frequently used. The reason for choosing the ethanol injection technique in the study is the absence of high-pressure processes that could disrupt the optical properties and structural conditions of the agents. The need to remove the residual ethanol remaining in the system after synthesis is one of the disadvantages of this technique. The residual ethanol can exert a solvent effect on the liposome membrane, disrupting vesicle stability. Throughout the study process, a rotary evaporator was used to remove the ethanol from the membrane. In the liposomes synthesized via this method, AuNPs reside in the aqueous core of the liposome, while BODIPY molecules integrate into the lipid bilayer. The time-dependent signals of the synthesized liposomes were monitored through periodic photoacoustic measurements on the 1st, 7th, and 14th days. In this context, the restrictive effect of the cholesterol amount on thermoelastic expansion was examined to find the ideal balance between leakage resistance and signal amplitude (at a 25% cholesterol ratio) and to demonstrate that higher ratios (35%) lead to acoustic damping. The study was conducted in two main stages and in two different laboratories: the synthesis of liposomal contrast agents and the performance of in vitro photoacoustic imaging analyses. In the first stage, liposomes were successfully synthesized using the necessary chemical reagents, while in the second stage, photoacoustic signal measurements of the synthesized products were acquired and analyzed in detail. In line with the findings obtained at the end of the study, the tight localization of the BODIPY agent within the lipid membrane triggered the aggregation-caused quenching mechanism according to Kasha's rule, thereby suppressing fluorescent emission and consequently converting the energy entirely into heat; this allowed the BODIPY dye to generate a stronger photoacoustic signal compared to the AuNPs. As a result of the periodic measurements, photoacoustic signals with high deviation were obtained in liposome formulations containing 0% mol cholesterol due to the generation of a high leakage variance. In liposomes with 35% mol cholesterol in their membrane, it was observed that thermoelastic expansion was severely suppressed due to excessive membrane rigidity, and consequently, a significant drop in the photoacoustic signal occurred. In light of all these data, a 25% cholesterol ratio was determined as the optimum threshold value that both ensures formulation stability and minimizes acoustic damping. These optimized nanocarriers offer a strong foundation for the development of sensitive theranostic platforms intended for future clinical applications.

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