Lazer Ablasyonu ile Sentezlenmiş Levan Kaplı Altın Nanoparçacıkların siRNA Transfeksiyon Ajanı Olma Potansiyeli Üstüne Çalışma
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Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Kırıkkale Üniversitesi
Access Rights
info:eu-repo/semantics/openAccess
Abstract
Bu çalışmada, altın nanoparçacık (AuNP) temelli kısa interferanslı RNA (siRNA) transfeksiyon vektörü tasarlanmıştır. Sıvı içinde atımlı lazer ablasyon yöntemiyle (PLAL) levan polisakkariti içeren ortamda sentezlenen AuNP’ler (Levan-AuNP), karakterizasyon testlerinin ardından MCF-7 meme kanseri hücrelerinde siRNA transfeksiyon çalışmalarında kullanılmıştır. Hedefsiz siRNA sekansı ile gerçekleşen %48’lik Levan-AuNP-siRNA etkileşimi A260 absorbans ölçümleriyle bulunmuştur. Farklı derişimlerde Levan-AuNP ve 50 nM siGLO Green transfeksiyon indikatörü hazırlanarak MCF-7 hücrelerindeki optimum siRNA transfeksiyon koşulları belirlenmiştir. Transfeksiyon çalışmalarında öncelikle üçlü floresan boyama (siGLO Green, propidyum iyodür (PI) ve Hoechst 33342) yapılmış ve sitotoksisite görülmemiştir. Özellikle en yüksek derişimdeki Levan-AuNP grubunda ilk günde transfeksiyon gerçekleşirken, tek başına siGLO Green grubunda transfeksiyon gerçekleşmemiştir. 2 ve 4 günlük inkübasyonda ise tüm gruplarda transfeksiyon gözlemlenmiştir. PI ve Hoechst kullanılmadan yapılan ikinci transfeksiyon çalışmalarında ise siGLO Green floresan şiddetinin belirgin şekilde arttığı ve özellikle Levan-AuNP-siGLO Green komplekslerinde daha kuvvetli ve noktalı bir floresan ışıma görülmüştür. Ortalama floresan şiddetleri ölçümlerine göre, tüm Levan-AuNP gruplarında istatistiksel açıdan anlamlı ölçüde artış bulunmuştur. Sonuç olarak, bu çalışmada geliştirilen Levan-AuNP’nin, mevcut transfeksiyon ajanlarına alternatif olabileceği gösterilmiştir.
Here, gold nanoparticle (AuNP)-based small interfering RNA (siRNA) vector was developed. AuNPs synthesized by pulsed laser ablation in liquid (PLAL) method in a medium containing levan polysaccharide (Levan-AuNP) were characterized, then utilized in siRNA transfection studies on MCF-7 breast cancer cells. The interaction between non-targeted siRNA sequence and Levan-AuNP-siRNA was found to be 48% via A260 absorbance measurements. Optimal conditions for siRNA transfection in MCF-7 cells were determined by preparing different concentrations of Levan-AuNP and 50 nM siGLO Green transfection indicator. In transfection studies, initially triple fluorescent staining protocols (siGLO Green, propidium iodide (PI), and Hoechst 33342) were conducted and no cytotoxicity was observed. In 1 day-incubation, the transfection was realized especially in the highest concentration Levan-AuNP group while it was unsuccessful in siGLO Green group alone. Transfection was observed in all groups after 2 and 4 days of incubation. In the secondary transfection studies without PI and Hoechst, the fluorescence intensity of siGLO Green increased significantly, and stronger and punctuate fluorescence emission was seen in Levan-AuNP-siGLO Green complexes. Regarding the mean fluorescence intensity measurements, a statistical significance was confirmed in all Levan-AuNP groups. As a whole, the developed Levan-AuNP could be proposed as alternatives to the existing transfection agents.
Here, gold nanoparticle (AuNP)-based small interfering RNA (siRNA) vector was developed. AuNPs synthesized by pulsed laser ablation in liquid (PLAL) method in a medium containing levan polysaccharide (Levan-AuNP) were characterized, then utilized in siRNA transfection studies on MCF-7 breast cancer cells. The interaction between non-targeted siRNA sequence and Levan-AuNP-siRNA was found to be 48% via A260 absorbance measurements. Optimal conditions for siRNA transfection in MCF-7 cells were determined by preparing different concentrations of Levan-AuNP and 50 nM siGLO Green transfection indicator. In transfection studies, initially triple fluorescent staining protocols (siGLO Green, propidium iodide (PI), and Hoechst 33342) were conducted and no cytotoxicity was observed. In 1 day-incubation, the transfection was realized especially in the highest concentration Levan-AuNP group while it was unsuccessful in siGLO Green group alone. Transfection was observed in all groups after 2 and 4 days of incubation. In the secondary transfection studies without PI and Hoechst, the fluorescence intensity of siGLO Green increased significantly, and stronger and punctuate fluorescence emission was seen in Levan-AuNP-siGLO Green complexes. Regarding the mean fluorescence intensity measurements, a statistical significance was confirmed in all Levan-AuNP groups. As a whole, the developed Levan-AuNP could be proposed as alternatives to the existing transfection agents.
Description
Keywords
Levan, altın nanoparçacık, lazer, siRNA, transfeksiyon, Levan, gold nanoparticle, laser, siRNA, transfection, Engineering
Journal or Series
Uluslararası Mühendislik Araştırma ve Geliştirme Dergisi
WoS Q Value
Scopus Q Value
Volume
1
Issue
1-282
