Glioblastoma tedavisinde kombin ilaç yüklü katmanlı nanolif yüzeylerin tasarımı ve kullanılabilirliğinin analizi
Loading...
Date
2024-08-20
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Bursa Uludağ Üniversitesi
Abstract
Glioblastoma (GB), tüm beyin tümörlerinin %12-15'ini ve astrositomların %50-60'ını oluşturan yetişkinlerde en sık görülen ve en agresif beyin tümörüdür. Cerrahi rezeksiyon, eş zamanlı verilen kemoterapi ilacı olan Temozolomid (TMZ) ve radyasyondan oluşan mevcut standart tedaviden sonra hastaların medyan sağkalımı sadece 12.6 aydır. GB’lerde çoğu durumda tümörün tam rezeksiyonu mümkün olmadığından sıklıkla kısa süre içerisinde nüks görülür. Son yıllarda yapılan çalışmalar ile birlikte geliştirilen yeni teknolojiler çeşitli kanser türlerinde onkolojik tedavinin başarısını artırsa da GB’de sistemik toksisite, kan-beyin bariyerinden (BBB) sınırlı penetrasyon, tümör bölgesinde yeterli ilaç konsantrasyona ulaşamama ve bu tedavilerin kısa yarı ömrü GB tedavisi için geliştirilen yaklaşımların başarısını sınırlamaktadır. Bu nedenle GB tedavisinde tümör yatağında kanser karşıtı ajanların kontrollü salım ile birlikte lokal etkisine odaklanan tedavi yaklaşımlarına ihtiyaç duyulmaktadır. Güncel çalışmalar, TMZ'nin GB tümör yatağına lokal olarak verilmesinin, TMZ'nin sistemik dolaşımını önleyerek ve normal dokuları ilacın toksisitesinden koruyarak, sistemik tedavide sıklıkla gözlenen klinik yan etkileri azalttığını kanıtlamıştır. Ayrıca, intrakraniyal tümörler için lokal tedavinin öncülerinden biri olan karmustin (BCNU) yüklü Gliadel ® gofretleri, tümör yatağında içerisinde yüklü olan ajanın lokal olarak kontrollü bir şekilde salmasını sağlamak için biyouyumlu polimer teknolojisi olarak geliştirilmiş ve FDA tarafından onaylanmıştır. Ancak malzemenin sertliği, büyüklüğü ve aşırı difüzyonu nedeniyle görülen yan etkiler bu ilacın kullanımını sınırlandırmıştır. Üstelik, BCNU glial tümörler de dahil olmak üzere birçok kanserin tedavisinde kullanılabilmesine rağmen, güncel çalışmalar BCNU’nun GB için sadece TMZ ile birlikte bir takviye olarak kullanılmasını önermektedir ve bu kapsamda TMZ tabanlı tedavi hala GB hastaları için standart tedavi olmaya devam etmektedir. Bu nedenle, TMZ ve TMZ’nin etkinliğini arttırabilecek biyoaktif bileşik kullanan polimer tabanlı kontrollü salım sistemleri araştırmacıların tümör yatağındaki GB'nin lokal tedavisi için kullanılabilecek hedeflerden biri haline gelmiştir. Bunun sebebi farklı moleküler mekanizmaları hedef alan terapötik ajanların kombinasyonuyla oluşan multimodal terapötik yaklaşımlarının, GB hücreleri üzerinde mevcut kemoterapi ilaçlarının tek kullanımından daha etkili bir sitotoksik etkiye sahip olmasıdır. Çalışmalar, kemoterapötiklerin aktivitesinin, anti-oksidan özelliklere sahip bazı biyoaktif bileşiklerle birlikte uygulandıklarında kemoterapi direncini ortadan kaldırabileceğini göstermektedir. Biyoaktif bileşenler arasında yer alan fenollerin ve flavonoidlerin, fenolik hidroksil gruplarının redoks özellikleri ve kimyasal yapılarındaki yapısal ilişkiler nedeniyle serbest radikallere karşı önemli anti-oksidan aktiviteler sağladığı kanıtlanmıştır. Zeytin ağacı yaprak özütleri (Olea europaea leaf extratct; OLE) ve içerisindeki biyoaktif bileşikler (Oleuropein; OL, hidroksitirozol; HT, tirozol; TYR ve rutin) kanser hücrelerinin çoğalmasını azaltan ve onların agresifliğini baskılayan anti-oksidan özelliklere sahiptirler. Ancak bu bileşiklerin zayıf sulu çözünürlük, düşük absorpsiyon ve moleküllerin hızlı metabolizması, onları klinik uygulaması için ciddi anlamda sınırlandırmaktadır. İlaç dağıtım sistemleri, farmakolojik uygulama sürecinde belirli maddeler/ilaçlar için taşıyıcı olarak işlev görecek şekilde küçük moleküller veya makromoleküllerle yüklenebilen nanomalzemeleri içerir. Polimerler arasında, elektrospinning ile üretilen nanolifler, yüksek yükleme kapasitesi, yüksek kapsülleme değişimi ve büyük yüzey alanı-hacim oranı gibi vazgeçilmez özellikler nedeniyle cerrahi rezeksiyondan sonra tümör yatağına implante edilebilir ilaç taşıyıcı sistemleri olarak geliştirilmiştir. Nanolifler, ayarlanabilir ilaç oranlarının kontrollü ve uzun süreli salınmasını sağlayarak tümörler üzerinde istenilen etkinin oluşmasını sağlarlar. Biyouyumluluğu, biyolojik olarak parçalanabilirliği ve iyi mekanik özellikleri nedeniyle ilaçların kapsüllenmesini sağlayan poli(laktik asit) (PLA) gibi çeşitli doğal ve sentetik polimerler elektrospinning ile üretilebilmektedir. Hidrofobik bir polimer olan PLA, biyolojik çevreyle temas ettiğinde genellikle hidroliz yoluyla laktik asit veya karbondioksit ve suya ayrışmaya başlar. PLA, vücutta iyi tolere edilir ve zamanla güvenli bozunma ürünlerine ayrışır. Bu nedenle, son çalışmalar PLA'nın sitotoksisite olmadan tümör ve normal hücreler tarafından içselleştirildiğini ve genellikle iyi tolere edildiğini göstermektedir. Ancak, ilaç/polimer karışım ile elektrospinning üretimi sırasında, nanoliflerin patlama salım göstermeleri nedeniyle ilacın nanoliflerin yüzeyinden hızla salınması meydana gelir. Çekirdek-kabuk elektrospinning, ilaç moleküllerinin salımını dikte ederek ilacı polimerlere başlangıçta patlama salım olmadan kapsülleyerek bu sınırlamanın üstesinden gelir ve bu da daha yüksek yükleme süreçleriyle elde edilebilen kontrollü bir salımla sonuçlanır. Bu kapsamda mevcut tezde GB tümörlerinin rezeksiyondan sonra tümör alanında lokal tedavi olarak kullanılabilecek, kontrollü salım yeteneği ile cerrahi rezeksiyon sonrası kalan hücrelerin proliferasyonunu ve agresifliğini baskılayabilecek biyouyumlu biyoaktif bileşik ve kemoterapi ilacı yüklü hibrit katmanlı kompozit nanolif ağ (LHN) tasarlandı. Bu LHN yüzey, biyoaktif bileşik ve/veya ilaç yüklenmiş iki nanolif katmanından oluşmaktadır. LHN’yi oluşturan ilk katman, polivinil alkol (PVA) nanolifi, tümör rezeksiyonundan sonra kalan kanser hücrelerini öldürmek için ilacı/biyoaktif bileşiği anında salması için tasarlandı. LHN’yi oluşturan ikinci katman, çekirdek-kabuk PLA nanolif ağı, bu ajanları uzun vadede ve tümör büyümesini önlemek için kontrollü bir şekilde serbest bırakmak üzere tasarlandı. Ek olarak, bu katmanlar arasına ilaç/biyoaktif bileşik eklenmesiyle iki katman arasının desteklenmesi sağlandı. Mevcut doktora tez çalışmasının ilk bölümünde, PLA ve LHN nanomalzeme içerisine yüklenecek olan biyoaktif molekülün belirlenebilmesi amacıyla, OLE ve fenolikleri olan, OL, HT, TYR ve rutinin apoptoz teşvik edici ve kanser hücrelerini baskılama üzerine etkilerinin hem tek başına ve hem de TMZ ile kombinasyon halinde GB hücrelerine karşı araştırılması ve karşılaştırılması amaçlanmıştır. Bu kapsamda biyoaktif bileşiklerin ayrı ayrı ve TMZ ile kombinasyon halinde GB hücre hatları olan T98G ve A172 hücrelerine etkileri, gerçek zamanlı hücre çoğalma analizi ve WST-1 analiziyle, hücre döngüsü dağılımı ve hücre içi oksidatif stres durumu kit protokolü kullanarak Muse hücre analiz sistemiyle, apoptozu teşvik edici özelliği çift akridin oranj/propidyum iyodür (AO/PI) boyama ve Annexin V analiziyle gerçekleştirildi. Sonrasında biyoaktif bileşiklerin tek başlarına ve TMZ ile kombinasyon halinde GB tümör agresifliğini baskılama yönündeki etkilerinin belirlenmesi için, çizik oluşumuyla yara iyileşme analizi, koloni oluşum analizi, GB tümör sferlerinin canlılık analizi ve GB kök benzeri hücre (GSC) analizi gerçekleştirildi. Tez çalışmasının bu bölümündeki bulgulara göre, OLE'deki aktif fenolik bileşikler GB hücre proliferasyonunu inhibe etti ve bu bileşikler hücre döngüsünü G2/M aşamasında tutuklayarak koloni oluşumunu azalttı. OLE ve içerisindeki fenolik bileşikler sinerji skoruna göre TMZ’ye katkılı etki gösterdiği belirlendi. OL'ün apoptoz etkisi iki hücre hattında da tek başına TMZ tedavisinden daha yüksekti (p<0,0001) ve tüm OLE fenolikleri arasında TMZ+OL kombinasyonu en yüksek apoptotik etki gösterdi. Ek olarak, tek başına HT, T98G hücrelerinde tek başına TMZ'den daha yüksek bir apoptotik etki gösterdi (p<0,0001). TYR ve rutin ise, tedavi edilmemiş hücrelere kıyasla her iki hücre hattında da apoptozu indükledi. OL, GB tümör sfer boyutunu ve canlılığını en çok bastırdı (T98G'de: 4,1 kat, p<0,0001; A172'de: 2,5 kat, p<0,0001). TMZ+OL kombinasyonu, tümör sfer büyümesinde önemli bir azalmaya yol açtı ve dahası tek başına kemoterapi tedavisindeki tümör sferlerine kıyasla hipoksik çekirdek bölgesini azalttı. TYR ve rutin OL ile benzer bir etki gösterdi ve T98G ve A172 hücrelerinde tümör sfer boyutlarını sırasıyla 2 ve 1,7 kat azalttı (p<0,0001). T98G hücrelerinde TMZ+rutin kombinasyonunun GB tümör sferinin büyüklüğünün inhibisyonu üzerindeki etkisi, TMZ+HT ve TMZ+TYR'nin etkisine benzerdi. Tedavi edilmemiş tümör sferlerine kıyasla OLE ve OLE fenolikleri, sferin çekirdek bölgesinde hipoksi kaynaklı nekroz oluşumunu azalttı. OL ve HT, CD133 ve OCT4 RNA ifadelerini azaltmada OLE’ye benzerdi. Buna karşılık, TYR ve rutin, tedavi edilmeyen hücrelere kıyasla CD133 ve OCT4'ün RNA seviyelerinin ifadesini azaltmış olsa da bu baskılama seviyesi iki hücre tipine göre değişkenlik gösterdi. TMZ+rutin’in kombin tedavisi, A172 hücrelerinde CD133’ün RNA seviyesini, her iki hücre hattında da OCT4’ün RNA seviyelerini önemli derecede baskıladı (p<0,05). TMZ tedavisi tek başına T98G hücrelerinin reaktif oksijen türlerinin (ROS) üretimini etkilememesine karşın, OLE, OL ve rutin tedavisi, tedavi edilmemiş T98G ve A172 hücrelerine kıyasla ROS üretimini azalttı (p<0,0001). Dahası, TMZ ve OLE fenoliklerinin TMZ ile kombini ROS miktarını önemli ölçüde azalttı. OLE fenoliklerinin her biri tek başına ve TMZ ile kombin halde her iki hücre hattında da yara iyileşme hızını yavaşlattı. Sonuç olarak tez çalışmasının bu bölümündeki veriler, OL ve rutinin GB hücrelerinin tedavisine karşı yeni bir terapötik ajan olabileceğini ve TMZ tedavisinin etkinliğini artırmada umut vaat ettiğini gösterdi. Bu sebeple tez çalışmasının bir sonraki bölümünde analiz edilecek olan PLA nanolifin içerisine OL ve rutin kapsüllenmesine karar verildi. Mevcut doktora tez çalışmasının ikinci bölümünde, GB lokal tedavisinde kullanılmak üzere geliştirilen LHN’nin ikinci katmanını oluşturan çekirdek-kabuk PLA nanolif ağlarının üretilmesi, bu nanoliflere OL, rutin ve TMZ kapsüllenmesi ve bu nanoliflerden yüklenen bileşiklerin kontrollü salımlarının oluşturulmasıyla GB hücrelerinin çoğalmalarını ve agresifliğini baskılamalarının araştırılması amaçlanmıştır. Elektrospinning işlemi ile üretilen PLA çekirdek-kabuk nanolif ağlarının cm2’sine OL, rutin ve TMZ’nin yarı maksimum inhibitör konsantrasyonları (IC50) kapsüllenerek (PLAOL, PLArutin ve PLATMZ) morfolojileri taramalı elektron mikroskobu (SEM) ile görselleştirildi ve toplam daldırma yöntemi ile PLA ağlarının salım özellikleri belirlendi. Hücre çoğalması için gerçek zamanlı hücre izleme analizi, hücre canlılığı için çift AO/PI boyaması, göç kapasitesi için çizik yara iyileşme analizi ve tümör sfer canlılığı için sfer oluşumu testi kullanıldı. Tez çalışmasının bu bölümündeki bulgulara göre, PLArutin ve PLATMZ nanolif ağlarının düzgün, yoğun, homojen, pürüzsüz ve boncuksuz morfolojiye sahip olduğu gözlemlenirken, PLAOL nanolif ağlarında liflerin yapıştığı ve boncuk benzeri morfolojiye sahip olduğu gözlemlendi. Tüm yüklü PLA nanolif ağları, 133±30,7–139±20,5 nm arasında ortalama çapa sahip çekirdek-kabuk yapılarına sahipti. PLAOL'ün salım değeri 6. ve 24. saatleri hariç zamanla artış gösterdi. PLAOL'ün maksimum salımı 72. saatte (~2,74 ppm), minimum salma değeri ise sırasıyla 6. ve 24. saatte 0,65 ppm ve 0,51 ppm oldu. 48 saatte ortalama molekül salımı 18 saatten düşük olsa da standart sapmalar dikkate alındığında, bu salma profilleri her iki dönem için de benzerdi. Genel olarak, 18. ve 48. saatte salınan moleküller benzer görünümdeydi. PLArutin’in ilaç salım değeri zamana bağlı olarak azalış gösterdi. PLArutin 18. saatte en yüksek salım değeri gösterirken, 24. saatte en düşük salım değeri gösterdi. PLArutin’in 1. ve 6. saatte stabil salım değerine yol açması rutinin moleküler yapısından kaynaklanıyordu. PLATMZ'den TMZ salımı zamana bağlı bir şekilde artış gösterdi. PLATMZ'nin maksimum molekül salımı 48. saatte (~7,25 ppm) olurken, minimum molekül salım değeri ise 1. saatte (1,36 ppm) gerçekleşti. Yüklü PLA nanoliflerinden moleküllerin yaklaşık %60'ının salımı 72 saatte olduğundan yüklü PLA ağları, besiyeri ortamında (CM) bu süre içerisinde salıma bırakıldı. PLAOL 24h-CM'de yetiştirilen T98G hücrelerinin canlılığı, tedavi edilmeyen hücrelere kıyasla %52,9'a düştü (p<0,0001). PLAOL’de 120 saate kadar CM’lerde yetiştirilen hücrelerde büyüme hızında doğrusal bir azalma gözlemlendi ve hücre canlılığı 120. saatin sonunda %13,6'ya düştü (p<0,0001). Buna karşılık, PLArutin 24h-CM T98G hücrelerinin çoğalma hızında ani bir düşüşe yol açarken (%8,1; p<0,0001), 120. saate kadar hücre büyümesinin seyri değişmedi. Bu bulgular, PLAOL ve PLArutin salımlarının hücre çoğalma testi ile benzer olduğunu doğruladı. Buna paralel olarak, PLATMZ+OL120h-CM ve PLATMZ+rutin120h-CM kombin tedaviler, T98G hücrelerinin çoğalma hızında tek başına tedavi olan PLATMZ 120h-CM'ye kıyasla daha fazla bir azalmaya yol açtı (PLATMZ+OL 120h-CM'de T98G hücre büyümesi: %1,4, p<0,0001; PLATMZ+rutin120h-CM'de: %1, p<0,0001; tedavi edilmeyen hücreler ile karşılaştırıldığında). PLAOL, PLArutin ve PLATMZ tedavileri, T98G hücrelerinde apoptoz belirteci olan at nalı biçimli çekirdeğe yol açtıkları görüldü ve apoptotik hücrelerde sırasıyla %62, %75 ve %28'inde nükleer parçalanma gözlendi. OL'ün TMZ üzerindeki katkısal etkisini destekleyerek kombin tedavide apoptotik morfolojiye sahip hücre sayısı PLATMZ+OL tedavilerinden sonra daha da arttı. Bulgular bir önceki bölümde görülen tek başına OL, rutin ve TMZ'nin hücre morfolojisi üzerindeki etkileri ile PLA nanolifine yüklendiği haliyle benzerdi. PLAOL ve PLArutin, T98G hücrelerinin yara iyileşme oranını azalttı ve yara alanı oranları sırasıyla %70,6 (p<0,0001) ve %79,3 (p<0,0001) idi. PLArutin ile tedavi edilen hücrelerde, PLAOL ile tedavi edilen hücrelere kıyasla yara alanının daha geniş olduğu ve bu kapsamda migrasyonu daha çok baskıladığı tespit edildi. Beklendiği gibi, kombin etki olan PLATMZ+OL ve PLATMZ+rutin ile tedavinin, tek başına tedavi olan PLATMZ ile karşılaştırıldığında T98G hücrelerinin yara iyileşme oranını daha çok azalttığı görüldü (p<0,0001). Ayrıca, PLAOL ve PLArutin in-vitro GB invazyon modelinde T98G hücre invazyonunu baskıladı. PLAOL, PLArutin ve PLATMZ, tedavi edilmemiş sferlere kıyasla T98G sfer boyutunu ve canlılığını azalttı. OL'ün TMZ üzerindeki katkılı etkisi ile sferlerin yapısı bozuldu ve tümör sferlerinde canlılık anlamlı derecede azaldı (p<0,0001). Ölü hücrelerin bolluğu PLArutin ile tedavi edilen GB hücrelerinde de oldukça fazlaydı. Rutin aracılı canlılığın azalması ayrıca PLATMZ+rutin ile tedavi edilen T98G sferlerinde de gözlendi; burada canlılık tek başına tedavi olan PLATMZ muamelesindeki sferlerden önemli ölçüde daha düşüktü (p<0,0001). Sonuç olarak tez çalışmasının bu bölümündeki veriler, OL ve rutin yüklü çekirdek-kabuk PLA nanoliflerinin tekrarlayan GB hücrelerine karşı moleküllerin kontrollü ve yavaş salımı ile yeni ve etkili bir terapötik araç olabileceğini göstermektedir. Ayrıca sonuçlar değerlendirildiğinde, tez çalışmasının bir sonraki bölümünde GB lokal tedavisi için geliştirilen LHN içerisine rutin bileşiğinin yüklenmesine karar verildi. Mevcut doktora tez çalışmasının üçüncü bölümünde ise GB'nin lokal tedavisi için farklı salım özelliklerine sahip, canlı sistemde kullanımı uygun ve biyouyumlu yenilikçi nanomateryal olan katmanlı hibrit kompozit nanolif oluşturan LHN tasarlandı. LHN’yi oluşturan ilk katman, içerisine kapsüllenen TMZ ve rutini anında salması için PVA nanolifinden oluşmaktadır. Bu yüzey tümör rezeksiyonundan sonra kalan kanser hücrelerini kısa süreli salım ile anında etki etmesi için tasarlandı. LHN’yi oluşturan ikinci katman, tümör büyümesini baskılamak için bu ajanların kontrollü uzun vadeli salınması yapması için çekirdek-kabuk PLA nanolif yüzeyden oluşmaktadır. Ek olarak, bu nanolifler arasına TMZ ve rutin püskürtülmesiyle iki katman desteklendi. Tezin bu bölümünde tasarlanan TMZ ve/veya rutin yüklü LHN’lerin (LHNTMZ, LHNrutin, LHNTMZ+rutin), in-vitro analizler ile GB hücrelerinin agresifliği üzerindeki etkilerinin değerlendirilmesi, etkilenen onkolojik süreçler ve ilişkili protein ağlarının belirlenmesi amaçlandı. Sonrasında ise LHNTMZ, LHNrutin ve LHNTMZ+rutin’in GB tümör boyutuna etkisi ve sistemik yan etki riski ile inflamatuar yanıt üzerindeki etkileri in-vivo ortotopik GB modeli kullanılarak belirlenmesi hedeflendi. LHN’ler elektrospinning ile üretildikten sonra nanolif ağlarının çapları ve yüzey morfolojileri SEM ile görüntülendi ve in-vitro salımı UV-Vis-NIR spektrofotometresi ile ölçüldü. LHN ağlarının GB hücreleri üzerindeki etkisini analiz etmek amacıyla, hücre büyümesi için gerçek zamanlı hücre izleme analizi, apoptoz durumu için AO/PI boyama ve Annexin V analizi, mitokondriyal membran potansiyeli testi (Δψm), migrasyon analizi için yara-çizik analizi, invazyon analizi için mikroakışkan istila testi IC-çip analizi, anjiyogenez analizi için HUVEC tüp oluşum testi, koloni oluşturma testi ve 3 boyutlu (3B) sfer canlılığı için bir sfer oluşumu testi kullanıldı. Yüklü nanoliflerin epitelyal-mezenkimal geçiş (EMT), GSC büyümesi ve LncRNA ifadelerine etkisi RT-PCR analiziyle incelendi. Proteomiks analizi ile, protein ağı netleştirildi. LHN'nin uygulanabilirliği ve kullanılabilirliği, ortotopik C6 GB sıçan modeli kullanılarak test edildi. Manyetik rezonans görüntüleme ve immünohistokimyasal (IHC) analiz tümörün varlığını doğruladı. Tümörlerin mitokondriyal yapısı transmisyon elektron mikroskobu (TEM) ile değerlendirildi ve PARP1 ifadesi analiz edildi. Serum aspartat aminotransferaz (AST), alanin aminotransferaz (ALT), kreatin, üre düzeyleri ve splenosit IFNg/IL4 oranı LHN’lerin biyogüvenliğini kanıtladı. Tez çalışmasının bu bölümündeki bulgulara göre, yüklü LHN’lerin iç katmanları dolgu formunda görülürken, üst ve alt katmanları lifli bir formda yapılandırıldığı görüldü. LHNrutin ağlarının ortalama lif çapı 89–253 nm aralığında 164,1±38 nm olduğu belirlendi. Rutin içeren lifler düz bir yapıyken, TMZ lifi çıkıntılı yapıdaydı. LHNTMZ ağlarındaki bu çıkıntılar, 89-485 nm arasında değişkenlik gösterdi. LHNrutin'den rutin salımı, PVA ve aktif reaktif biriktirilmiş katmanların çözünmesi nedeniyle ilk 6 saat boyunca patlama şeklinde salım görüldü. Rutin salımının minimum değeri 1. saatte (0,586±0,26 ppm) gözlenirken, maksimum salım değeri 6. saatte görüldü (3,44±0,11 ppm). Devamında 168. saate kadar kontrollü salım başladı. LHNTMZ'den TMZ’nin salımı kademeli olarak artarak LHNTMZ'den madde salımı maksimum 168. saatte (0,66±0,28 ppm) tespit edilirken, minimum salım 18. saatte (8,05±0,67 ppm) tespit edildi. Devamında 168 saatte ise salım miktarının arttığı görüldü. LHNTMZ, LHNrutin ve LHNTMZ+rutin GB hücre proliferasyonunu 96. saate kadar inhibe etti. Ek olarak LHNTMZ+rutin’in kombin etkisi T98G hücre çoğalmasının baskılanmasında, LHNTMZ'nin etkisinden daha önemliydi (p<0,0001). LHNrutin ve LHNTMZ+rutin tedavi edilmemiş T98G hücrelerine kıyasla Δψm'yi azalttı (sırasıyla; p=0,0058, p=0,003). LHNrutin ve LHNTMZ+rutin’in kombin tedavisi, EMT ve GSC RNA ifadelerini ve LncRNA’ları inhibe ederek GB hücrelerindeki 2D migrasyon, 3B invazyonu ve koloni oluşumunu baskıladı. LHNrutin tedavisi, tedavi Ayrıca, LHNTMZ+rutin tedavisinin, tedavi edilmeyen hücrelere karşı invazyonda 51,5±1,41 katlık bir azalmaya sebep olduğu görüldü (p<0,0001). LHNrutin ve kombin tedavi edilen HUVEC hücrelerinde kılcal benzeri tüp ağlarının oluşumu önemli ölçüde azaldı (p<0,0001). LHNTMZ+rutin kombinasyonu tümör sfer boyutunu, tedavi edilmeyen ve tek başına LHNTMZ uygulanan hücrelere kıyasla önemli ölçüde azalttı (p<0,0001). Tüm yüklü LHN’ler GB hücrelerinde apoptozu teşvik etti. Proteomiks analizler, mitokondri, endoplazmik retikulum (ER) ve golgi proteinlerinde önemli değişiklikler olduğunu ve yüklü LHN’lerin tümör agresifliğini baskıladığını gösterdi. LHNTMZ'nin tek başına hücre proteomu üzerinde LHNrutin ve LHNTMZ+rutin'den daha düşük bir etkisi oldu ve ayrıca kombinasyon tedavisinin daha etkili olduğunu gösterdi. Ortotopik C6 kaynaklı GB sıçan modelinde tedavi edilmeyen sıçanların, sağ frontal lobunun derin beyaz maddesinde infiltratif ödemle birlikte tümörün beynin diğer bölgelerine yayıldığı tespit edildi. LHNTMZ ve LHNrutin ile tedavi edilen sıçanlarda tümör boyutu tedavi edilmeyen sıçanlara göre daha küçüktü ve infiltratif ödem gözlenmedi. Hematoksilen-eozin (H&E) boyama ile belirlenen tümör hacimlerine göre, tedavi edilmemiş GB sıçanlarındaki tümör boyutu 382±65,7 mm3 iken, LHNTMZ'den sonra 69±21 mm3, LHNrutin'den sonra 88,5±23,33 mm3 ve LHNTMZ+rutin’den sonra 11±9 mm3 olduğu tespit edildi. Tümör mitokondrilerindeki yapısal değişiklikler, azalmış membran potansiyeli ve azalmış PARP ifadesi, tümör hücrelerinde apoptotik yolların aktivasyonunu gösterdi ve bu, tümör hücrelerinin mitotik aktivitesinin azaldığını gösteren fosfo-histon H3 (PHH3)'teki azalma ile bir kez daha doğrulandı. Ek olarak, GB modelinde LHN'lerin lokal uygulanması, yan doku iltihabına veya olumsuz sistemik etkilere neden olmadan GB tümörünün agresif özelliklerini hafifletti. Yüklü LHN’lerin kullanılabilirliği ve biyouyumluluğu anjiyogenez belirteci CD31'deki azalma, serebellumun H&E boyamasında iltihap veya nekrozun yokluğu, IFN-γ üretiminin artması, dalak T hücrelerinde IL-4 seviyelerinin azalması ve daha düşük serum AST seviyeleri ile kanıtlandı. Sonuç olarak tez çalışmasının bu bölümündeki veriler, tümör bölgesine lokal olarak uygulandığında LHNTMZ ve LHNrutin’in GB sıçan modelinde GB tümörlerinin agresif özellikleri üzerindeki hafifletici etkisini vurguladı. Özellikle, LHNTMZ+rutin’in tümör azaltıcı etkisi, LHNTMZ ile karşılaştırıldığında kayda değerdi. Bu nedenle, kontrollü ve uzun salım yaparak rezeksiyon alanındaki tümör hücrelerinin çoğalmasını engelleyen bir tedavi yaklaşımı olarak LHNTMZ+rutin’nin kullanılabilirliği oldukça dikkat çekicidir. LHNTMZ, LHNrutin ve LHNTMZ+rutin’in lokal doku inflamasyonuna neden olmaması ve karaciğer ve böbrek üzerindeki yan etkilere sebep olmaması üretilen materyalin kullanılabilirliğini desteklemektedir. Bu nedenle, LHNTMZ, LHNrutin ve LHNTMZ+rutin GB tümör bölgesine uygulanması canlı sistem için uygun olarak kabul edildi. Bulgularımız toplu olarak LHNTMZ+rutin’in GB'nin lokal tedavisi için yenilikçi bir biyouyumlu nano yaklaşım olduğunu gösterdi. Bulgularımız ilaç/ilaç adayı yüklü LHN’lerin GB'nin lokal tedavisi için umut verici biyouyumlu bir model olduğunu göstermektedir. Bu LHN’lerin GB tedavisinde nüksü engellemede kullanılabilecek ekonomik bir ürün olabileceği belirlendi.
Glioblastoma (GB) is the most common and aggressive brain tumor in adults, accounting for 12-15% of all brain tumors and 50-60% of astrocytomas. The median survival of patients after current standard treatment, which consists of surgical resection, the concurrent chemotherapy drug Temozolomide (TMZ), and radiation, is only 12.6 months. Since complete resection of the tumor is not possible in most cases of GB, recurrence often occurs in a short time. Although new technologies developed with the studies conducted in recent years increase the success of oncological treatment in various types of cancer, systemic toxicity in GB, limited penetration through the blood-brain barrier (BBB), inability to reach sufficient drug concentration in the tumor area, and the short half-life of these treatments limit the success of GB treatment approaches. Therefore, treatment approaches that focus on the local effect of anti-cancer agents with controlled release in the tumor bed are needed in the treatment of GB. Current studies have proven that local delivery of TMZ to the GB tumor bed reduces clinical side effects often observed with systemic therapy by preventing systemic circulation of TMZ and protecting normal tissues from the drug's toxicity. In addition, Gliadel ® wafers loaded with carmustine (BCNU), one of the pioneers of local treatment for intracranial tumors, were developed and approved by the FDA as a biocompatible polymer technology to provide locally controlled release of the loaded agent in the tumor bed. However, side effects due to the hardness, size, and excessive diffusion of the material have limited the use of this drug. Moreover, although BCNU can be used to treat many cancers, including glial tumors, current studies recommend using it only as a supplement together with TMZ for GB. To this extent, TMZ-based therapy remains the standard treatment for GB patients. Therefore, polymer-based controlled release systems using TMZ and bioactive compounds that can increase the effectiveness of TMZ have become one of the targets that researchers can use for local treatment of GB in the tumor bed. This is because multimodal therapeutic approaches, consisting of the combination of therapeutic agents targeting different molecular mechanisms, have a more effective cytotoxic effect on GB cells than the single use of existing chemotherapy drugs. Studies show that the activity of chemotherapeutics can eliminate chemotherapy resistance when administered together with certain bioactive compounds with anti-oxidant properties. It has been proven that phenols and flavonoids, among the bioactive components, provide important anti-oxidant activities against free radicals due to the redox properties of phenolic hydroxyl groups and the structural relationships in their chemical structures. Olive tree leaf extracts (Olea europaea leaf extract; OLE) and the bioactive compounds (Oleuropein; OL, hydroxytyrosol; HT, tyrosol; TYR and rutin) have anti-oxidant properties that reduce the proliferation of cancer cells and suppress their aggressiveness. However, the poor aqueous solubility, low absorption, and rapid metabolism of the molecules of these compounds seriously limit them for clinical application. Drug delivery systems include nanomaterials that can be loaded with small molecules or macromolecules to act as carriers for specific substances/drugs in pharmacological administration. Among polymers, nanofibers produced by electrospinning have been developed as implantable drug carrier systems in the tumor bed after surgical resection due to indispensable properties such as high loading capacity, high encapsulation variation, and large surface area-to-volume ratio. Nanofibers ensure the desired effect on tumors by providing controlled and long-term release of adjustable drug rates. Various natural and synthetic polymers, such as poly(lactic acid) (PLA), which enable the encapsulation of drugs due to their biocompatibility, biodegradability, and good mechanical properties, can be produced by electrospinning. PLA, a hydrophobic polymer, begins to decompose into lactic acid, carbon dioxide, and water when it comes into contact with the biological environment, usually through hydrolysis. PLA is well tolerated in the body and breaks down into safe degradation products over time. Therefore, recent studies show that PLA is internalized by tumor and normal cells without cytotoxicity and is generally well tolerated. However, during electrospinning production with the drug/polymer mixture, the drug's rapid release from the nanofibers' surface occurs due to the burst release of the nanofibers. Core-shell electrospinning overcomes this limitation by dictating the release of drug molecules and encapsulating the drug into polymers without initial burst release, resulting in a controlled release that can be achieved through higher loading processes. In this context, the current thesis, a hybrid layered composite nanofiber network (LHN) loaded with biocompatible, bioactive compounds and chemotherapy drugs was designed, which can be used as a local treatment in the tumor area after resection of GB tumors and can suppress the proliferation and aggressiveness of remaining cells after surgical resection with its controlled release ability. This LHN surface consists of two layers of nanofibers loaded with bioactive compounds and/or drugs. The first layer forming the LHN, polyvinyl alcohol (PVA) nanofiber, was designed to instantly release the drug/bioactive compound to kill remaining cancer cells after tumor resection. The second layer, the core-shell PLA nanofiber network that forms the LHN, is designed to release these agents in a controlled manner over the long term and to prevent tumor growth. Additionally, support between the two layers was achieved by adding drugs/bioactive compounds between these layers. The first part of the current doctoral thesis study aimed to investigate and compare the effects of OLE and its phenolics, OL, HT, TYR, and rutin, on apoptosis-promoting and suppressing cancer cells against GB cells, both alone and in combination with TMZ. In this context, it will be possible to determine the bioactive molecule that will be loaded into PLA and LHN nanomaterials. In this context, the effects of bioactive compounds individually and in combination with TMZ on T98G and A172 cells, which are GB cell lines, were evaluated by real-time cell proliferation analysis and WST-1 analysis, cell cycle distribution and intracellular oxidative stress status by Muse cell analysis system using the kit protocol and apoptosis-promoting properties were determined by double acridine orange/propidium iodide (AO/PI) staining and Annexin V analysis. Subsequently, to determine the effects of bioactive compounds alone and in combination with TMZ on suppressing GB tumor aggressiveness, wound healing analysis by scratch formation, colony formation analysis, viability analysis of GB tumor spheres, and GB stem-like cell (GSC) analysis were performed. According to the findings in this part of the thesis study, the active phenolic compounds in OLE inhibited GB cell proliferation. These compounds reduced colony formation by arresting the cell cycle in the G2/M phase. According to the synergy score, OLE and its phenolic compounds had an additive effect on TMZ. The apoptosis effect of OL was higher than TMZ treatment alone in both cell lines (p<0,0001), and among all OLE phenolics, the TMZ+OL combination showed the highest apoptotic effect. In addition, HT-only showed a higher apoptotic effect than TMZ-only in T98G cells (p<0,0001). However TYR and rutin induced apoptosis in both cell lines compared to untreated cells. OL most suppressed GB tumor sphere size and viability (in T98G: 4,1-fold, p<0,0001; in A172: 2,5-fold, p<0,0001). The TMZ+OL combination led to a significant reduction in tumor sphere growth and further reduced the hypoxic core area compared to tumor spheres in chemotherapy treatment alone. TYR and rutin showed a similar effect to OL and reduced tumor sphere sizes by 2- and 1,7-fold in T98G and A172 cells, respectively (p<0,0001). The effect of the TMZ+rutin combination on the inhibition of GB tumor sphere size in T98G cells was similar to the effect of TMZ+HT and TMZ+TYR. Compared to untreated tumor spheroids, OLE and OLE phenolics reduced hypoxia-induced necrosis in the core region of the sphere. OL and HT were similar to OLE in reducing CD133 and OCT4 RNA expressions. In contrast, TYR and rutin reduced the expression of RNA levels of CD133 and OCT4 compared to untreated cells, although this level of suppression varied between the two cell types. Combination treatment of TMZ+rutin significantly suppressed the RNA level of CD133 in A172 cells and the RNA levels of OCT4 in both cell lines (p<0,05). Although TMZ-only treatment did not affect reactive oxygen species (ROS) production of T98G cells, OLE, OL, and rutin treatment reduced ROS production compared to untreated T98G and A172 cells (p<0,0001). Moreover, the combination of TMZ and OLE phenolics with TMZ significantly reduced the amount of ROS. Each OLE phenolic alone and combined with TMZ slowed the wound healing rate in both cell lines. In conclusion, the data in this part of the thesis showed that OL and rutin may be new therapeutic agents against the treatment of GB cells and are promising in increasing the effectiveness of TMZ treatment. For this reason, it was decided to encapsulate OL and rutin into the PLA nanofiber, which will be analyzed in the next part of the thesis. The second part of the current doctoral thesis study aims to produce core-shell PLA nanofiber networks that form the second layer of LHN developed for use in the local treatment of GB and encapsulate OL, rutin, and TMZ into these nanofibers. In addition, it aims to investigate whether they suppress the proliferation and aggressiveness of GB cells by creating controlled releases of compounds loaded from these nanofibers. In the second part of the current doctoral thesis study, half maximum inhibitory concentrations (IC50) of OL, rutin, and TMZ were encapsulated (PLAOL, PLArutin, and PLATMZ) into the cm2 of PLA core-shell nanofiber networks produced by GB local electrospinning process. The morphologies of the nanofibers were visualized by scanning electron microscopy (SEM), and the release properties of PLA networks were determined using the total immersion method. Real-time cell tracking analysis was used for cell proliferation, double AO/PI staining for cell viability, scratch wound healing assay for migration capacity, and sphere formation assay for tumor sphere viability. According to the findings in this part of the thesis, it was observed that PLArutin and PLATMZ nanofiber networks had a uniform, dense, homogeneous, smooth, and bead-free morphology, while it was observed that the fibers in PLAOL nanofiber networks adhered and had a bead-like morphology. All loaded PLA nanofiber networks had core-shell structures with average diameters between 133±30,7–139±20,5 nm. PLAOL's emission value increased over time, except for the 6 and 24h. The maximum release of PLAOL was at the 72h (~2,74 ppm), while the minimum release value was 0,65 ppm and 0,51 ppm at the 6h and 24h, respectively. Although the average molecule release at 48h was lower than at 18h, these release profiles were similar for both periods when standard deviations were considered. In general, the molecules released at 18h and 48h appeared similarly. The drug release value of PLArutin decreased over time. PLArutin showed the highest release value at 18h and the lowest release value at 24h. The stable release value of PLArutin at 1st and 6h was due to the molecular structure of rutin. TMZ released by PLATMZ increased in a time-dependent manner. While the maximum molecule release of PLATMZ occurred at the 48h (~7,25 ppm), the minimum molecule release value occurred at the 1h (1,36 ppm). Since the release of approximately 60% of the molecules from the loaded PLA nanofibers was observed in 72h, the loaded PLA networks were released in the medium (CM) during this period. The viability of T98G cells grown in PLAOL 24h-CM decreased to 52,9% compared to untreated cells (p<0,0001). In PLAOL, a linear decrease in growth rate was observed in cells grown in CMs for up to 120h, and cell viability decreased to 13,6% at the end of the 120h (p<0,0001). In contrast, PLArutin caused a sudden decrease in the proliferation rate of 24h-CM T98G cells (8,1%; p<0,0001), while the course of cell growth did not change until the 120h. These findings confirmed that PLAOL and PLArutin releases were similar by cell proliferation assay. In parallel, the combined treatments PLATMZ+OL120h-CM and PLATMZ+rutin120h-CM led to a greater reduction in the proliferation rate of T98G cells compared to the treatment PLATMZ 120h-CM (T98G cells in PLATMZ+OL 120h-CM growth: 1,4%, p<0,0001; in PLATMZ+rutin120h-CM: 1%, p<0,0001; compared to untreated cells). PLAOL, PLArutin, and PLATMZ treatments appeared to induce a horseshoe-shaped nucleus, a marker of apoptosis, in T98G cells, and nuclear fragmentation was observed in 62%, 75%, and 28% of apoptotic cells, respectively. Supporting the additive effect of OL on TMZ, the number of cells with apoptotic morphology in the combined treatment further increased after PLATMZ+OL treatments. The findings were similar to the effects of OL, rutin, and TMZ as loaded onto PLA nanofiber on cell morphology, as seen in the previous section. PLAOL and PLArutin reduced the wound healing rate of T98G cells, and the wound area rates were 70,6% (p<0,0001) and 79,3% (p<0,0001), respectively. It was determined that the wound area was more significant in cells treated with PLArutin than in cells treated with PLAOL, and in this context, migration was suppressed more. As expected, treatment with the combined effect of PLATMZ+OL and PLATMZ+rutin reduced the wound healing rate of T98G cells more than PLATMZ (p<0,0001). Additionally, PLAOL and PLArutin suppressed T98G cell invasion in the in-vitro GB invasion model. PLAOL, PLArutin, and PLATMZ reduced T98G sphere size and viability compared to untreated spheres. With the additive effect of OL on TMZ, the structure of the spheres was disrupted, and the viability of the tumor spheres was significantly reduced (p<0,0001). The abundance of dead cells was relatively high in PLArutin-treated GB cells. Rutin-mediated viability reduction was also observed in PLATMZ+rutin-treated T98G spheres, where viability was significantly lower than in spheres in the PLATMZ treatment (p<0,0001). In conclusion, the data in this part of the thesis show that OL and rutin-loaded core-shell PLA nanofibers can be a new and effective therapeutic tool with the controlled and slow release of molecules against recurrent GB cells. Additionally, when the results were evaluated, it was decided to load the rutin compound into the LHN developed for the local treatment of GB in the next part of the thesis study. In the third part of the current doctoral thesis study, LHN, which forms a layered hybrid composite nanofiber, which is an innovative nanomaterial that has different release properties, is suitable for use in living systems, and is biocompatible was designed for the local treatment of GB. The first layer forming the LHN consists of TMZ encapsulated inside and PVA nanofiber to instantly release the rutin. Thanks to this surface, it was designed to instantly affect the remaining cancer cells after tumor resection with short-term release. The second layer forming the LHN consists of a core-shell PLA nanofiber surface for controlled long-term release of these agents to suppress tumor growth. Additionally, two layers were supported by sputtering TMZ and rutin between these nanofibers. In this part of the thesis, it was aimed to evaluate the effects of designed TMZ and/or rutin-loaded LHNs (LHNTMZ, LHNrutin, LHNTMZ+rutin) on the aggressiveness of GB cells through in-vitro analysis and to determine the affected oncological processes and associated protein networks. Afterward, it was aimed to determine the effects of LHNTMZ, LHNrutin, and LHNTMZ+rutin on GB tumor size, systemic side effect risk, and inflammatory response using an in-vivo orthotopic GB model. After LHNs were produced by electrospinning, the diameters and surface morphologies of the nanofiber networks were visualized by SEM, and the in-vitro release was measured by a UV-Vis-NIR spectrophotometer. To analyze the effect of LHN networks on GB cells, real-time cell tracking analysis for cell growth, AO/PI staining and Annexin V analysis for apoptosis status, mitochondrial membrane potential assay (Δψm), wound-scratch analysis for migration analysis, microfluidic invasion assay IC-chip analysis for invasion analysis, HUVEC tube formation assay for angiogenesis analysis, colony formation assay, and a sphere formation assay for 3-dimensional (3D) sphere viability were used. The effects of loaded nanofibers on epithelial-mesenchymal transition (EMT), GSC growth, and LncRNA expressions were examined by RT-PCR analysis. By proteomics analysis, the protein network was clarified. The feasibility and usability of LHN were tested using the orthotopic C6 GB rat model. Magnetic resonance imaging and immunohistochemical (IHC) analysis confirmed the presence of the tumor. The mitochondrial structure of the tumors was evaluated by transmission electron microscopy (TEM), and PARP1 expression was analyzed. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine, urea levels, and splenocyte IFNg/IL4 ratio proved the biosafety of LHNs. According to the findings in this part of the thesis, while the inner layers of the loaded LHNs were seen as filling, the upper and lower layers were structured in a fibrous form. The average fiber diameter of LHNrutin networks was determined to be 164,1±38 nm in the range of 89–253 nm. While the fibers containing rutin had a flat structure, the TMZ fiber had a protruding structure. These protrusions in LHNTMZ networks varied between 89-485 nm. Rutin release from LHNrutin showed burst release during the first 6h due to the dissolution of PVA and active reagent deposited layers. The minimum value of rutin release was observed at the 1h (0,586±0,26 ppm), while the maximum release was observed at the 6h (3,44±0,11 ppm). Afterward, controlled release started until the 168h. The release of TMZ from LHNTMZ gradually increased. While the maximum release of substance from LHNTMZ was detected at the 168h (0,66±0,28 ppm), the minimum release was detected at the 18h (8,05±0,67 ppm). It was observed that the amount of emission increased in the following 168h. LHNTMZ, LHNrutin, and LHNTMZ+rutin inhibited GB cell proliferation until the 96h. In addition, the combined effect of LHNTMZ+rutin was more significant than that of LHNTMZ in suppressing T98G cell proliferation (p<0,0001). LHNrutin and LHNTMZ+rutin reduced Δψm compared to untreated T98G cells (p=0,0058, p=0,003, respectively). Combination treatment of LHNrutin and LHNTMZ+rutin suppressed 2D migration, 3D invasion, and colony formation in GB cells by inhibiting EMT and GSC RNA expressions and LncRNA genes. LHNrutin treatment led to an 11,4±1,5-fold reduction in invasion compared to untreated cells (p<0,0001). Additionally, it was observed that LHNTMZ+rutin treatment caused a 51,5±1,41-fold decrease in an invasion against untreated cells (p<0,0001). The combination of LHNrutin and TMZ significantly reduced the formation of capillary-like tube networks in treated HUVEC cells (p<0,0001). The combination of LHNTMZ+rutin significantly reduced tumor sphere size compared to untreated cells and cells treated with LHNTMZ (p<0,0001). All loaded LHNs promoted apoptosis in GB cells. Proteomics analyses showed significant changes in mitochondria, endoplasmic reticulum (ER), and Golgi proteins, and loaded LHNs suppressed tumor aggressiveness. LHNTMZ had a lower effect on the cell proteome than LHNrutin and LHNTMZ+rutin, indicating that the combination treatment was more effective. In the orthotopic C6-induced GB rat model, it was determined that the tumor spread to other parts of the brain with infiltrative edema in the deep white matter of the right frontal lobe of untreated rats. Tumor size in rats treated with LHNTMZ and LHNrutin was smaller than in untreated rats, and no infiltrative edema was observed. According to tumor volumes determined by hematoxylin-eosin (H&E) staining, the tumor size in untreated GB rats was 382±65,7 mm3. It was determined to be 69±21 mm3 with LHNTMZ, 88,5±23,33 mm3 with LHNrutin, and 11±9 mm3 with LHNTMZ+rutin. Structural changes in tumor mitochondria, decreased membrane potential, and decreased PARP expression indicated activation of apoptotic pathways in tumor cells, and this was once again confirmed by the decrease in phospho-histone H3 (PHH3), indicating decreased mitotic activity of tumor cells. In addition, the local application of LHNs in the GB model alleviated the aggressive features of the GB tumor without causing collateral tissue inflammation or adverse systemic effects. The availability and biocompatibility of loaded LHNs was evidenced by the decrease in the angiogenesis marker CD31, the absence of inflammation or necrosis on H&E staining of the cerebellum, increased production of IFN-γ, decreased levels of IL-4 in spleen T cells, and lower serum AST levels. In conclusion, the data in this part of the thesis highlighted the attenuating effect of LHNTMZ and LHNrutin on the aggressive features of GB tumors in the GB rat model when applied locally to the tumor site. In particular, the tumor-reducing effect of LHNTMZ+rutin was remarkable compared to LHNTMZ. Therefore, LHNTMZ+rutin is remarkable as a treatment approach that prevents the proliferation of tumor cells in the resection area by providing a controlled and extended-release. The fact that LHNTMZ, LHNrutin, and LHNTMZ+rutin do not cause local tissue inflammation or cause side effects on the liver and kidney supports that the produced material can be used. Therefore, applying LHNTMZ, LHNrutin, and LHNTMZ+rutin to the GB tumor site was considered suitable for the live system. Our findings demonstrated that LHNTMZ+rutin is a promising biocompatible innovative nano approach for the local treatment of GB. Our findings indicate that drug/drug candidate-loaded LHNs are a promising biocompatible model for the local treatment of GB. It was determined that these LHNs could be an economical product that can be used to prevent recurrence in the treatment of GB.
Glioblastoma (GB) is the most common and aggressive brain tumor in adults, accounting for 12-15% of all brain tumors and 50-60% of astrocytomas. The median survival of patients after current standard treatment, which consists of surgical resection, the concurrent chemotherapy drug Temozolomide (TMZ), and radiation, is only 12.6 months. Since complete resection of the tumor is not possible in most cases of GB, recurrence often occurs in a short time. Although new technologies developed with the studies conducted in recent years increase the success of oncological treatment in various types of cancer, systemic toxicity in GB, limited penetration through the blood-brain barrier (BBB), inability to reach sufficient drug concentration in the tumor area, and the short half-life of these treatments limit the success of GB treatment approaches. Therefore, treatment approaches that focus on the local effect of anti-cancer agents with controlled release in the tumor bed are needed in the treatment of GB. Current studies have proven that local delivery of TMZ to the GB tumor bed reduces clinical side effects often observed with systemic therapy by preventing systemic circulation of TMZ and protecting normal tissues from the drug's toxicity. In addition, Gliadel ® wafers loaded with carmustine (BCNU), one of the pioneers of local treatment for intracranial tumors, were developed and approved by the FDA as a biocompatible polymer technology to provide locally controlled release of the loaded agent in the tumor bed. However, side effects due to the hardness, size, and excessive diffusion of the material have limited the use of this drug. Moreover, although BCNU can be used to treat many cancers, including glial tumors, current studies recommend using it only as a supplement together with TMZ for GB. To this extent, TMZ-based therapy remains the standard treatment for GB patients. Therefore, polymer-based controlled release systems using TMZ and bioactive compounds that can increase the effectiveness of TMZ have become one of the targets that researchers can use for local treatment of GB in the tumor bed. This is because multimodal therapeutic approaches, consisting of the combination of therapeutic agents targeting different molecular mechanisms, have a more effective cytotoxic effect on GB cells than the single use of existing chemotherapy drugs. Studies show that the activity of chemotherapeutics can eliminate chemotherapy resistance when administered together with certain bioactive compounds with anti-oxidant properties. It has been proven that phenols and flavonoids, among the bioactive components, provide important anti-oxidant activities against free radicals due to the redox properties of phenolic hydroxyl groups and the structural relationships in their chemical structures. Olive tree leaf extracts (Olea europaea leaf extract; OLE) and the bioactive compounds (Oleuropein; OL, hydroxytyrosol; HT, tyrosol; TYR and rutin) have anti-oxidant properties that reduce the proliferation of cancer cells and suppress their aggressiveness. However, the poor aqueous solubility, low absorption, and rapid metabolism of the molecules of these compounds seriously limit them for clinical application. Drug delivery systems include nanomaterials that can be loaded with small molecules or macromolecules to act as carriers for specific substances/drugs in pharmacological administration. Among polymers, nanofibers produced by electrospinning have been developed as implantable drug carrier systems in the tumor bed after surgical resection due to indispensable properties such as high loading capacity, high encapsulation variation, and large surface area-to-volume ratio. Nanofibers ensure the desired effect on tumors by providing controlled and long-term release of adjustable drug rates. Various natural and synthetic polymers, such as poly(lactic acid) (PLA), which enable the encapsulation of drugs due to their biocompatibility, biodegradability, and good mechanical properties, can be produced by electrospinning. PLA, a hydrophobic polymer, begins to decompose into lactic acid, carbon dioxide, and water when it comes into contact with the biological environment, usually through hydrolysis. PLA is well tolerated in the body and breaks down into safe degradation products over time. Therefore, recent studies show that PLA is internalized by tumor and normal cells without cytotoxicity and is generally well tolerated. However, during electrospinning production with the drug/polymer mixture, the drug's rapid release from the nanofibers' surface occurs due to the burst release of the nanofibers. Core-shell electrospinning overcomes this limitation by dictating the release of drug molecules and encapsulating the drug into polymers without initial burst release, resulting in a controlled release that can be achieved through higher loading processes. In this context, the current thesis, a hybrid layered composite nanofiber network (LHN) loaded with biocompatible, bioactive compounds and chemotherapy drugs was designed, which can be used as a local treatment in the tumor area after resection of GB tumors and can suppress the proliferation and aggressiveness of remaining cells after surgical resection with its controlled release ability. This LHN surface consists of two layers of nanofibers loaded with bioactive compounds and/or drugs. The first layer forming the LHN, polyvinyl alcohol (PVA) nanofiber, was designed to instantly release the drug/bioactive compound to kill remaining cancer cells after tumor resection. The second layer, the core-shell PLA nanofiber network that forms the LHN, is designed to release these agents in a controlled manner over the long term and to prevent tumor growth. Additionally, support between the two layers was achieved by adding drugs/bioactive compounds between these layers. The first part of the current doctoral thesis study aimed to investigate and compare the effects of OLE and its phenolics, OL, HT, TYR, and rutin, on apoptosis-promoting and suppressing cancer cells against GB cells, both alone and in combination with TMZ. In this context, it will be possible to determine the bioactive molecule that will be loaded into PLA and LHN nanomaterials. In this context, the effects of bioactive compounds individually and in combination with TMZ on T98G and A172 cells, which are GB cell lines, were evaluated by real-time cell proliferation analysis and WST-1 analysis, cell cycle distribution and intracellular oxidative stress status by Muse cell analysis system using the kit protocol and apoptosis-promoting properties were determined by double acridine orange/propidium iodide (AO/PI) staining and Annexin V analysis. Subsequently, to determine the effects of bioactive compounds alone and in combination with TMZ on suppressing GB tumor aggressiveness, wound healing analysis by scratch formation, colony formation analysis, viability analysis of GB tumor spheres, and GB stem-like cell (GSC) analysis were performed. According to the findings in this part of the thesis study, the active phenolic compounds in OLE inhibited GB cell proliferation. These compounds reduced colony formation by arresting the cell cycle in the G2/M phase. According to the synergy score, OLE and its phenolic compounds had an additive effect on TMZ. The apoptosis effect of OL was higher than TMZ treatment alone in both cell lines (p<0,0001), and among all OLE phenolics, the TMZ+OL combination showed the highest apoptotic effect. In addition, HT-only showed a higher apoptotic effect than TMZ-only in T98G cells (p<0,0001). However TYR and rutin induced apoptosis in both cell lines compared to untreated cells. OL most suppressed GB tumor sphere size and viability (in T98G: 4,1-fold, p<0,0001; in A172: 2,5-fold, p<0,0001). The TMZ+OL combination led to a significant reduction in tumor sphere growth and further reduced the hypoxic core area compared to tumor spheres in chemotherapy treatment alone. TYR and rutin showed a similar effect to OL and reduced tumor sphere sizes by 2- and 1,7-fold in T98G and A172 cells, respectively (p<0,0001). The effect of the TMZ+rutin combination on the inhibition of GB tumor sphere size in T98G cells was similar to the effect of TMZ+HT and TMZ+TYR. Compared to untreated tumor spheroids, OLE and OLE phenolics reduced hypoxia-induced necrosis in the core region of the sphere. OL and HT were similar to OLE in reducing CD133 and OCT4 RNA expressions. In contrast, TYR and rutin reduced the expression of RNA levels of CD133 and OCT4 compared to untreated cells, although this level of suppression varied between the two cell types. Combination treatment of TMZ+rutin significantly suppressed the RNA level of CD133 in A172 cells and the RNA levels of OCT4 in both cell lines (p<0,05). Although TMZ-only treatment did not affect reactive oxygen species (ROS) production of T98G cells, OLE, OL, and rutin treatment reduced ROS production compared to untreated T98G and A172 cells (p<0,0001). Moreover, the combination of TMZ and OLE phenolics with TMZ significantly reduced the amount of ROS. Each OLE phenolic alone and combined with TMZ slowed the wound healing rate in both cell lines. In conclusion, the data in this part of the thesis showed that OL and rutin may be new therapeutic agents against the treatment of GB cells and are promising in increasing the effectiveness of TMZ treatment. For this reason, it was decided to encapsulate OL and rutin into the PLA nanofiber, which will be analyzed in the next part of the thesis. The second part of the current doctoral thesis study aims to produce core-shell PLA nanofiber networks that form the second layer of LHN developed for use in the local treatment of GB and encapsulate OL, rutin, and TMZ into these nanofibers. In addition, it aims to investigate whether they suppress the proliferation and aggressiveness of GB cells by creating controlled releases of compounds loaded from these nanofibers. In the second part of the current doctoral thesis study, half maximum inhibitory concentrations (IC50) of OL, rutin, and TMZ were encapsulated (PLAOL, PLArutin, and PLATMZ) into the cm2 of PLA core-shell nanofiber networks produced by GB local electrospinning process. The morphologies of the nanofibers were visualized by scanning electron microscopy (SEM), and the release properties of PLA networks were determined using the total immersion method. Real-time cell tracking analysis was used for cell proliferation, double AO/PI staining for cell viability, scratch wound healing assay for migration capacity, and sphere formation assay for tumor sphere viability. According to the findings in this part of the thesis, it was observed that PLArutin and PLATMZ nanofiber networks had a uniform, dense, homogeneous, smooth, and bead-free morphology, while it was observed that the fibers in PLAOL nanofiber networks adhered and had a bead-like morphology. All loaded PLA nanofiber networks had core-shell structures with average diameters between 133±30,7–139±20,5 nm. PLAOL's emission value increased over time, except for the 6 and 24h. The maximum release of PLAOL was at the 72h (~2,74 ppm), while the minimum release value was 0,65 ppm and 0,51 ppm at the 6h and 24h, respectively. Although the average molecule release at 48h was lower than at 18h, these release profiles were similar for both periods when standard deviations were considered. In general, the molecules released at 18h and 48h appeared similarly. The drug release value of PLArutin decreased over time. PLArutin showed the highest release value at 18h and the lowest release value at 24h. The stable release value of PLArutin at 1st and 6h was due to the molecular structure of rutin. TMZ released by PLATMZ increased in a time-dependent manner. While the maximum molecule release of PLATMZ occurred at the 48h (~7,25 ppm), the minimum molecule release value occurred at the 1h (1,36 ppm). Since the release of approximately 60% of the molecules from the loaded PLA nanofibers was observed in 72h, the loaded PLA networks were released in the medium (CM) during this period. The viability of T98G cells grown in PLAOL 24h-CM decreased to 52,9% compared to untreated cells (p<0,0001). In PLAOL, a linear decrease in growth rate was observed in cells grown in CMs for up to 120h, and cell viability decreased to 13,6% at the end of the 120h (p<0,0001). In contrast, PLArutin caused a sudden decrease in the proliferation rate of 24h-CM T98G cells (8,1%; p<0,0001), while the course of cell growth did not change until the 120h. These findings confirmed that PLAOL and PLArutin releases were similar by cell proliferation assay. In parallel, the combined treatments PLATMZ+OL120h-CM and PLATMZ+rutin120h-CM led to a greater reduction in the proliferation rate of T98G cells compared to the treatment PLATMZ 120h-CM (T98G cells in PLATMZ+OL 120h-CM growth: 1,4%, p<0,0001; in PLATMZ+rutin120h-CM: 1%, p<0,0001; compared to untreated cells). PLAOL, PLArutin, and PLATMZ treatments appeared to induce a horseshoe-shaped nucleus, a marker of apoptosis, in T98G cells, and nuclear fragmentation was observed in 62%, 75%, and 28% of apoptotic cells, respectively. Supporting the additive effect of OL on TMZ, the number of cells with apoptotic morphology in the combined treatment further increased after PLATMZ+OL treatments. The findings were similar to the effects of OL, rutin, and TMZ as loaded onto PLA nanofiber on cell morphology, as seen in the previous section. PLAOL and PLArutin reduced the wound healing rate of T98G cells, and the wound area rates were 70,6% (p<0,0001) and 79,3% (p<0,0001), respectively. It was determined that the wound area was more significant in cells treated with PLArutin than in cells treated with PLAOL, and in this context, migration was suppressed more. As expected, treatment with the combined effect of PLATMZ+OL and PLATMZ+rutin reduced the wound healing rate of T98G cells more than PLATMZ (p<0,0001). Additionally, PLAOL and PLArutin suppressed T98G cell invasion in the in-vitro GB invasion model. PLAOL, PLArutin, and PLATMZ reduced T98G sphere size and viability compared to untreated spheres. With the additive effect of OL on TMZ, the structure of the spheres was disrupted, and the viability of the tumor spheres was significantly reduced (p<0,0001). The abundance of dead cells was relatively high in PLArutin-treated GB cells. Rutin-mediated viability reduction was also observed in PLATMZ+rutin-treated T98G spheres, where viability was significantly lower than in spheres in the PLATMZ treatment (p<0,0001). In conclusion, the data in this part of the thesis show that OL and rutin-loaded core-shell PLA nanofibers can be a new and effective therapeutic tool with the controlled and slow release of molecules against recurrent GB cells. Additionally, when the results were evaluated, it was decided to load the rutin compound into the LHN developed for the local treatment of GB in the next part of the thesis study. In the third part of the current doctoral thesis study, LHN, which forms a layered hybrid composite nanofiber, which is an innovative nanomaterial that has different release properties, is suitable for use in living systems, and is biocompatible was designed for the local treatment of GB. The first layer forming the LHN consists of TMZ encapsulated inside and PVA nanofiber to instantly release the rutin. Thanks to this surface, it was designed to instantly affect the remaining cancer cells after tumor resection with short-term release. The second layer forming the LHN consists of a core-shell PLA nanofiber surface for controlled long-term release of these agents to suppress tumor growth. Additionally, two layers were supported by sputtering TMZ and rutin between these nanofibers. In this part of the thesis, it was aimed to evaluate the effects of designed TMZ and/or rutin-loaded LHNs (LHNTMZ, LHNrutin, LHNTMZ+rutin) on the aggressiveness of GB cells through in-vitro analysis and to determine the affected oncological processes and associated protein networks. Afterward, it was aimed to determine the effects of LHNTMZ, LHNrutin, and LHNTMZ+rutin on GB tumor size, systemic side effect risk, and inflammatory response using an in-vivo orthotopic GB model. After LHNs were produced by electrospinning, the diameters and surface morphologies of the nanofiber networks were visualized by SEM, and the in-vitro release was measured by a UV-Vis-NIR spectrophotometer. To analyze the effect of LHN networks on GB cells, real-time cell tracking analysis for cell growth, AO/PI staining and Annexin V analysis for apoptosis status, mitochondrial membrane potential assay (Δψm), wound-scratch analysis for migration analysis, microfluidic invasion assay IC-chip analysis for invasion analysis, HUVEC tube formation assay for angiogenesis analysis, colony formation assay, and a sphere formation assay for 3-dimensional (3D) sphere viability were used. The effects of loaded nanofibers on epithelial-mesenchymal transition (EMT), GSC growth, and LncRNA expressions were examined by RT-PCR analysis. By proteomics analysis, the protein network was clarified. The feasibility and usability of LHN were tested using the orthotopic C6 GB rat model. Magnetic resonance imaging and immunohistochemical (IHC) analysis confirmed the presence of the tumor. The mitochondrial structure of the tumors was evaluated by transmission electron microscopy (TEM), and PARP1 expression was analyzed. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine, urea levels, and splenocyte IFNg/IL4 ratio proved the biosafety of LHNs. According to the findings in this part of the thesis, while the inner layers of the loaded LHNs were seen as filling, the upper and lower layers were structured in a fibrous form. The average fiber diameter of LHNrutin networks was determined to be 164,1±38 nm in the range of 89–253 nm. While the fibers containing rutin had a flat structure, the TMZ fiber had a protruding structure. These protrusions in LHNTMZ networks varied between 89-485 nm. Rutin release from LHNrutin showed burst release during the first 6h due to the dissolution of PVA and active reagent deposited layers. The minimum value of rutin release was observed at the 1h (0,586±0,26 ppm), while the maximum release was observed at the 6h (3,44±0,11 ppm). Afterward, controlled release started until the 168h. The release of TMZ from LHNTMZ gradually increased. While the maximum release of substance from LHNTMZ was detected at the 168h (0,66±0,28 ppm), the minimum release was detected at the 18h (8,05±0,67 ppm). It was observed that the amount of emission increased in the following 168h. LHNTMZ, LHNrutin, and LHNTMZ+rutin inhibited GB cell proliferation until the 96h. In addition, the combined effect of LHNTMZ+rutin was more significant than that of LHNTMZ in suppressing T98G cell proliferation (p<0,0001). LHNrutin and LHNTMZ+rutin reduced Δψm compared to untreated T98G cells (p=0,0058, p=0,003, respectively). Combination treatment of LHNrutin and LHNTMZ+rutin suppressed 2D migration, 3D invasion, and colony formation in GB cells by inhibiting EMT and GSC RNA expressions and LncRNA genes. LHNrutin treatment led to an 11,4±1,5-fold reduction in invasion compared to untreated cells (p<0,0001). Additionally, it was observed that LHNTMZ+rutin treatment caused a 51,5±1,41-fold decrease in an invasion against untreated cells (p<0,0001). The combination of LHNrutin and TMZ significantly reduced the formation of capillary-like tube networks in treated HUVEC cells (p<0,0001). The combination of LHNTMZ+rutin significantly reduced tumor sphere size compared to untreated cells and cells treated with LHNTMZ (p<0,0001). All loaded LHNs promoted apoptosis in GB cells. Proteomics analyses showed significant changes in mitochondria, endoplasmic reticulum (ER), and Golgi proteins, and loaded LHNs suppressed tumor aggressiveness. LHNTMZ had a lower effect on the cell proteome than LHNrutin and LHNTMZ+rutin, indicating that the combination treatment was more effective. In the orthotopic C6-induced GB rat model, it was determined that the tumor spread to other parts of the brain with infiltrative edema in the deep white matter of the right frontal lobe of untreated rats. Tumor size in rats treated with LHNTMZ and LHNrutin was smaller than in untreated rats, and no infiltrative edema was observed. According to tumor volumes determined by hematoxylin-eosin (H&E) staining, the tumor size in untreated GB rats was 382±65,7 mm3. It was determined to be 69±21 mm3 with LHNTMZ, 88,5±23,33 mm3 with LHNrutin, and 11±9 mm3 with LHNTMZ+rutin. Structural changes in tumor mitochondria, decreased membrane potential, and decreased PARP expression indicated activation of apoptotic pathways in tumor cells, and this was once again confirmed by the decrease in phospho-histone H3 (PHH3), indicating decreased mitotic activity of tumor cells. In addition, the local application of LHNs in the GB model alleviated the aggressive features of the GB tumor without causing collateral tissue inflammation or adverse systemic effects. The availability and biocompatibility of loaded LHNs was evidenced by the decrease in the angiogenesis marker CD31, the absence of inflammation or necrosis on H&E staining of the cerebellum, increased production of IFN-γ, decreased levels of IL-4 in spleen T cells, and lower serum AST levels. In conclusion, the data in this part of the thesis highlighted the attenuating effect of LHNTMZ and LHNrutin on the aggressive features of GB tumors in the GB rat model when applied locally to the tumor site. In particular, the tumor-reducing effect of LHNTMZ+rutin was remarkable compared to LHNTMZ. Therefore, LHNTMZ+rutin is remarkable as a treatment approach that prevents the proliferation of tumor cells in the resection area by providing a controlled and extended-release. The fact that LHNTMZ, LHNrutin, and LHNTMZ+rutin do not cause local tissue inflammation or cause side effects on the liver and kidney supports that the produced material can be used. Therefore, applying LHNTMZ, LHNrutin, and LHNTMZ+rutin to the GB tumor site was considered suitable for the live system. Our findings demonstrated that LHNTMZ+rutin is a promising biocompatible innovative nano approach for the local treatment of GB. Our findings indicate that drug/drug candidate-loaded LHNs are a promising biocompatible model for the local treatment of GB. It was determined that these LHNs could be an economical product that can be used to prevent recurrence in the treatment of GB.
Description
Keywords
Glioblastoma, Temozolomid, Rutin, Lokal tedavi, Hibrit katmanlı kompozit nanolif ağ, Temozolomide, Local treatment, Hybrid layered composite nanofiber webs