GSK2126458 loaded polycaprolactone (PCL)/ polyvinylpyrrolidone (PVP) electrospun membranes: Production, characterization and anticancer activity
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Tarih
2024
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Adana Alparslan Türkeş Bilim ve Teknoloji Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Deri kanseri, en tehlikeli kanser türlerinden biridir. Deri hücrelerinde oluşan DNA hasarının giderilememesiyle ortaya çıkan mutasyonlar sonucunda deride kanserli doku oluşumu görülebilmektedir. Deri kanseri insan vücudunda farklı bölgelere yayılma gösterebilen bir kanser türüdür ve iki ana gruba ayrılmaktadır: melanom ve melanom olmayan. Literatürdeki veriler melanom kanser türünün, deri kanseri vakalarının %1'ini oluşturduğunu belirtmektedir. Melanom deri kanserinin görülme sıklığı düşük olmasına rağmen hastaların ölüm oranları yüksektir. Deri kanseri tedavisinde farklı metotlar bulunmaktadır. Bu metotlardan en yaygın olanı biyopsi ve rezeksiyondur. Son yıllarda deri kanseri tedavisinde nanoteknoloji kullanımına ilgi artmıştır. Nanoteknoloji ile üretilen malzemeler molekül taşıyıcıları olarak kullanılmaktadır. Nanoteknolojik yöntemler kullanılarak üretilmiş membranlar ile ilacın spesifik bölgeye direkt uygulanabilmesi, daha hızlı tedavi ve azaltılmış yan etkiler elde edilmesi amaçlanmaktadır. Bu çalışmada, kanser ilacı olarak kullanılan GSK2126458 (Omipalisib®) ilacının elektroeğirme yöntemi ile elde edilen Polikaprolakton (PCL)/ Polivinilprolidon (PVP) fiber membranlar içerisine yüklenmesi ve melanom kanser hücre hattı üzerindeki etkilerinin incelenmesi amaçlanmıştır. Çalışma kapsamında elektroeğirme yöntemi ile GSK2126458 ilacını içeren, PCL/PVP/50 (50 µL ilaç ve 50 µL DMSO), PCL/PVP/75 (75 µL ilaç ve 25 µL DMSO) ve PCL/PVP/100 (100 µL ilaç) ve içermeyen PCL/PVP/C fiber membranlar üretilmiştir. Tüm örneklerde DMSO oranı sabit tutulmuştur. Üretilen fiber yapıların morfolojileri taramalı electron mikroskobu (SEM) ile kimyasal yapıları Fourier Transform Infrared Spectroscopy (FTIR) analizleri ile gerçekleştirilmiştir. Fiber çapları ve çap dağılımları SEM analizi ile elde edilen görüntülerden ImageJ program ile hesaplanmıştır. Fiber membranların su tutma özellikleri su temas açısı analizi ile mekanik özellikleri çekme-germe analizi ile belirlenmiştir. Üretilen fiber membranların antibakteriyel özelliklerinin belirlenmesi için E. coli, S. aureus, P. aeruginosa ve MRSA bakteri suşları üzerinde mikrobiyolojik analizler gerçekleştirilmiştir. Fiberlerin içerisine yüklenen GSK2126458 ilacının B16-F10 hücre hattı üzerindeki IC50 değeri resazurin analizi ile belirlenmiştir. İlacın fiber membrana eklendikten sonra melanom hücreleri üzerinde etkili olup olmadığı canlılık analizi, hücre morfolojisinin ve yayılma davranışının SEM analizi görüntülenmesi ve immünofloresan boyama ile araştırılmıştır. Tez çalışması sonucunda, GSK2126458 ilaç molekülü yüklenmiş ve ilaç içermeyen kontrol fiber membranlar düzgün ve boncuk içermeyen fiber yapısında elde edilmiştir. PCL/PVP/C, PCL/PVP/50, PCL/PVP/75 ve PCL/PVP/100 fiber gruplarının fiberlerinin ortlama çapları sırasıyla, 0.682?0,316, 0.588?0.240, 0.637?0.208 ve 0.611?0.196 µm olarak belirlenmiştir. PCL/PVP/C, PCL/PVP/50, PCL/PVP/75 ve PCL/PVP/100 fiber gruplarının su temas açısı analizleri sonucunda sırasıyla, 76.02?8.06, 83.63?1.75, 66.98?2.83 ve 82.92?12.82 dereceleri elde edilmiştir ve membranların hidrofilik yapıları gösterilmiştir. FTIR analizi ile PCL ve PVP polimerlerine spesifik pikler gösterilmiştir ancak ilaç molekülü için spesifik bir pik ilacın polimer yapısına gömülü olmasından dolayı elde edilememiştir. GSK2126458 ilacının B16-F10 fare melanom hücre hattı üzerinde IC50 değeri 49.21 µM olarak belirlenmiştir. İlaç yüklü membranlardaki hücre canlılığı ilaç içermeyen fiber membranlara göre analiz edildiğinde PCL/PVP/50, PCL/PVP/75 ve PCL/PVP/100 fiber membranlarda hücre canlılığının 24 saat için sırasıyla %72.25, %64.61 ve %57.82 (p<0.05) oldukları belirlenmiştir. 48 saat için ise sırasıyla, %74.76, %70.85 (p<0.05) ve %60.19 (p<0.01) oldukları belirlenmiştir. SEM görüntülerinde hücre yayılmasının kontrol grubunda diğer gruplara göre daha belirgin olduğu gösterilmiştir. Fiber membranlar üzerinde kültüre edilen hücrelerin fibröz aktin proteinin anti F-aktin antikoru ve çekirdeğinin DAPI ile boyanması ile elde edilen immünofloresan boyama görüntülerinde hücre yoğunluğunun artan ilaç molekülü miktarı ile azaldığı gösterilmiştir. Hücre çekirdeğinde belirgin değişiklikler görülmemiştir. Sonuç olarak, GSK2126458 ilacı yüklü PCL/PVP fiber membranların tez çalışması kapsamında başarı ile üretildiği ve B16-F10 fare melanom hücre hattı üzerinde etkili olduğu gösterilmiştir.
Skin cancer is one of the most dangerous cancers. The mutations resulting from the failure to repair DNA damage in skin cells result in the formation of cancer tissue in the skin. Skin cancer is a type of cancer that can spread to different parts of the human body and is divided into two main groups: melanoma and non-melanoma. The data in the literature indicate that the type of melanoma cancer accounts for 1% of cases of skin cancer. Although the incidence of melanoma skin cancer is low, patients have high mortality rates. There are different methods of treating skin cancer. The most common of these methods are biopsy and resection. In recent years, interest in the use of nanotechnology in the treatment of skin cancer has increased. Materials produced by nanotechnology are used as molecular carriers. Membranes produced using nanotechnological methods are intended to enable the drug to be applied directly to a specific area, to faster treatment and to reduce side effects. In this study, it was aimed to load GSK2126458 (Omipalisib®), which is used as a cancer drug, into Polycaprolactone (PCL)/ Polyvinylprolidone (PVP) fiber membranes obtained by electrospinning method and to investigate its effects on melanoma cancer cell line. Within the scope of the study, PCL/PVP/50 (50 µL drug and 50 µL DMSO), PCL/PVP/75 (75 µL drug and 25 µL DMSO), PCL/PVP/100 (100 µL drug) and PCL/PVP/C fiber membranes containing GSK2126458 drug and PCL/PVP/C fiber membranes without drug were produced by electrospinning method. DMSO ratio was kept constant in all samples. The morphology of the produced fiber structures were analyzed by scanning electron microscopy (SEM) and their chemical structures were analyzed by Fourier Transform Infrared Spectroscopy (FTIR). Fiber diameters and diameter distributions were calculated from the images obtained by SEM analysis using ImageJ software. Water retention properties of the fiber membranes were determined by water contact angle analysis and mechanical properties were determined by tensile analysis. Antibacterial properties of the fiber membranes were determined using E. coli, S. aureus, P. aeruginosa and MRSA bacterial strains. The IC50 value of the GSK2126458 on B16-F10 cell line was determined by resazurin assay. The effect of the drug on melanoma cells after being added to the fiber membrane was determined by viability analysis, SEM analysis of cell morphology and spreading behavior and immunofluorescent staining. As a result of the thesis study, GSK2126458 drug molecule loaded and drug-free control fiber membranes were obtained in a smooth and bead-free fiber structure. The average diameters of the fibers of PCL/PVP/C, PCL/PVP/50, PCL/PVP/75 and PCL/PVP/100 membranes were calculated as 0.682?0,316, 0.588?0.240, 0.637?0.208 ve 0.611?0.196 µm, respectively. Hydrophilic structures of the membranes were shown by water contact angles of 76.02?8.06, 83.63?1.75, 66.98?2.83 ve 82.92?12.82 degrees for PCL/PVP/C, PCL/PVP/50, PCL/PVP/75 and PCL/PVP/100 fiber membranes, respectively. FTIR analysis showed specific peaks for PCL and PVP polymers, but a specific peak for the drug molecule could not be obtained because the drug was embedded in the polymer structure. The IC50 value of GSK2126458 on B16-F10 cell line was determined as 49.21 µM. When cell viability in drug-loaded membranes was analyzed compared to drug-free fiber membranes, it was determined that cell viability in PCL/PVP/50, PCL/PVP/75 and PCL/PVP/100 fiber membranes were 72.25%, 64.61% and 57.82% (p<0.05) for 24 hours, respectively. The viabilities were changed to 74.76%, 70.85% (p<0.05) and 60.19% (p<0.01) for 48 h, respectively. SEM images showed that cell spreading was more prominent in the control group than in the other groups. Immunofluorescence staining images obtained by staining fibrous actin protein of cells cultured on fiber membranes with anti F-actin antibody and nuclei with DAPI showed that cell density decreased with increasing amount of drug molecule. No significant changes were observed in the cell nucleus. In conclusion, it was shown that GSK2126458 drug loaded PCL/PVP fiber membranes were successfully produced within the scope of the thesis study and were effective on B16-F10 mouse melanoma cell line.
Skin cancer is one of the most dangerous cancers. The mutations resulting from the failure to repair DNA damage in skin cells result in the formation of cancer tissue in the skin. Skin cancer is a type of cancer that can spread to different parts of the human body and is divided into two main groups: melanoma and non-melanoma. The data in the literature indicate that the type of melanoma cancer accounts for 1% of cases of skin cancer. Although the incidence of melanoma skin cancer is low, patients have high mortality rates. There are different methods of treating skin cancer. The most common of these methods are biopsy and resection. In recent years, interest in the use of nanotechnology in the treatment of skin cancer has increased. Materials produced by nanotechnology are used as molecular carriers. Membranes produced using nanotechnological methods are intended to enable the drug to be applied directly to a specific area, to faster treatment and to reduce side effects. In this study, it was aimed to load GSK2126458 (Omipalisib®), which is used as a cancer drug, into Polycaprolactone (PCL)/ Polyvinylprolidone (PVP) fiber membranes obtained by electrospinning method and to investigate its effects on melanoma cancer cell line. Within the scope of the study, PCL/PVP/50 (50 µL drug and 50 µL DMSO), PCL/PVP/75 (75 µL drug and 25 µL DMSO), PCL/PVP/100 (100 µL drug) and PCL/PVP/C fiber membranes containing GSK2126458 drug and PCL/PVP/C fiber membranes without drug were produced by electrospinning method. DMSO ratio was kept constant in all samples. The morphology of the produced fiber structures were analyzed by scanning electron microscopy (SEM) and their chemical structures were analyzed by Fourier Transform Infrared Spectroscopy (FTIR). Fiber diameters and diameter distributions were calculated from the images obtained by SEM analysis using ImageJ software. Water retention properties of the fiber membranes were determined by water contact angle analysis and mechanical properties were determined by tensile analysis. Antibacterial properties of the fiber membranes were determined using E. coli, S. aureus, P. aeruginosa and MRSA bacterial strains. The IC50 value of the GSK2126458 on B16-F10 cell line was determined by resazurin assay. The effect of the drug on melanoma cells after being added to the fiber membrane was determined by viability analysis, SEM analysis of cell morphology and spreading behavior and immunofluorescent staining. As a result of the thesis study, GSK2126458 drug molecule loaded and drug-free control fiber membranes were obtained in a smooth and bead-free fiber structure. The average diameters of the fibers of PCL/PVP/C, PCL/PVP/50, PCL/PVP/75 and PCL/PVP/100 membranes were calculated as 0.682?0,316, 0.588?0.240, 0.637?0.208 ve 0.611?0.196 µm, respectively. Hydrophilic structures of the membranes were shown by water contact angles of 76.02?8.06, 83.63?1.75, 66.98?2.83 ve 82.92?12.82 degrees for PCL/PVP/C, PCL/PVP/50, PCL/PVP/75 and PCL/PVP/100 fiber membranes, respectively. FTIR analysis showed specific peaks for PCL and PVP polymers, but a specific peak for the drug molecule could not be obtained because the drug was embedded in the polymer structure. The IC50 value of GSK2126458 on B16-F10 cell line was determined as 49.21 µM. When cell viability in drug-loaded membranes was analyzed compared to drug-free fiber membranes, it was determined that cell viability in PCL/PVP/50, PCL/PVP/75 and PCL/PVP/100 fiber membranes were 72.25%, 64.61% and 57.82% (p<0.05) for 24 hours, respectively. The viabilities were changed to 74.76%, 70.85% (p<0.05) and 60.19% (p<0.01) for 48 h, respectively. SEM images showed that cell spreading was more prominent in the control group than in the other groups. Immunofluorescence staining images obtained by staining fibrous actin protein of cells cultured on fiber membranes with anti F-actin antibody and nuclei with DAPI showed that cell density decreased with increasing amount of drug molecule. No significant changes were observed in the cell nucleus. In conclusion, it was shown that GSK2126458 drug loaded PCL/PVP fiber membranes were successfully produced within the scope of the thesis study and were effective on B16-F10 mouse melanoma cell line.
Açıklama
Lisansüstü Eğitim Enstitüsü, Biyomühendislik Ana Bilim Dalı, Biyomühendislik Bilim Dalı
Anahtar Kelimeler
Biyomühendislik, Bioengineering
