Publication: Antisens oligonükleotid taşıyan sistem geliştirilmesi ve in vitro tayini
Abstract
Antisens oligonükleotidler (AS ODN), hedef mRNA veya pre-mRNA’ya komplementer dizilişe sahip olan ve selektif hibridizasyon ile gen ekspresyonunu durduran, kısa, sentetik, tek zincirli DNA molekülleri olup; kanser, AIDS gibi pekçok hastalığın tedavisi için gelecek vaad etmektedirler. Ancak bu moleküllerin tedavide kullanılmalarında en büyük sorun; stabilitelerinin ve hücreye penetrasyonlarının düşük olması ve bu nedenle de uygun bir taşıyıcı sisteme ihtiyaç duyulmasıdır. Bu çalışmada katyonik bir polimer olan kitozanla AS ODN’lerin kompleks ve nanopartikül formları hazırlanmış; yüzey yükü, partikül büyüklüğü gibi fizikokimyasal özellikleri incelenmiş ve uygun formülasyonların in vitro hücre kültüründe antisens etkinlikleri araştırılmıştır. Yaklaşık 200 nm boyutunda oldukları belirlenen 1:20 ve 1:100 (AS ODN: Kitozan) kompleksler ile nanopartiküllerin antisens özellikleri, HeLa hücresinde pSV-Beta-Gal plazmidi tarafından kodlanan Beta-galaktozidaz ekspresyonunun AS ODN taşıyıcı sistemlerin verilmesinden sonraki inhibisyonu ile ölçülmüştür. Sonuçta, her iki formda da kitozan, AS ODN’leri enzimatik parçalanmadan koruyabilmiştir ancak nanopartiküllerde, AS ODN ile kitozan arasında çok güçlü elektrostatik bağların oluşması sonucunda AS ODN salınımı gerçekleşmemiştir ve antisens özelliğinin de düşük olduğu saptanmıştır. 1:100 (-/ +) komplekslerinde ise özellikle pDNA’nın transfeksiyonundan 24 saat sonra hücrelere AS ODN verilmesiyle yüksek antisens etkinlik saptanmıştır. Kitozanın özellikle fosforotiyoat modifikasyonlu AS ODN için iyi bir taşıyıcı olduğu görülmüştür. Bu çalışma, gelecekte kitozan polimeri ile yapılacak antisens oligonükleotid taşıyıcı sistem araştırmaları için temel oluşturmaktadır.
DEVELOPMENT OF A NOVEL DELIVERY SYSTEM FOR ANTISENSE OLIGONUCLEOTIDES AND ITS IN VITRO INVESTIGATION Antisense oligonucleotides (AS ODNs) are short, single-stranded, synthetic molecules that have complementary sequences of targeted mRNA or pre-mRNA by which the selective hybridization yields the inhibition of gene expression. These molecules are promising molecules in the treatment of diseases such as cancer, AIDS, etc. However, the major problems in using such molecules in the treatment of the diseases mentioned above, are lack of stability and poor penetration into the cell, therefore; a spesific carrier is needed. In this thesis, the complex and nanoparticle forms of AS ODNs were prepared by using a cationic polymer called chitosan. Afterwards, the physicochemical properties such as zeta-potential and particle size of these formulations were measured and the antisense activity of the appropriate formulations were investigated in vitro. The antisense activity of 1:20 and 1:100 (AS ODN: Chitosan) complexes and nanoparticles which have approximately 200 nm particle size, were measured based on the inhibition of Beta-galactosidase coded by pSV-Beta-Gal plasmid followed by the addition of antisense containing formulations into the HeLa cells. Eventually, both forms protected AS ODNs from potential enzymatic degredations. However, due to the strong electrostatic bonds between chitosan and AS ODN in nanoparticle formulations, AS ODN failed to be released and consequently, the antisense activity of nanoparticles were observed to be poor. In 1:100 (-/ +) complex formulations, high antisense activity was observed especially in the case of adding AS ODNs after 24 hours from pDNA transfection into the cell. As a result, chitosan can be considered as a suitable carrier especially for phosphorothioate modified AS ODNs. This study can form the basis for the forthcoming studies related with carrier systems of AS ODNs that will be done with chitosan polymer.
DEVELOPMENT OF A NOVEL DELIVERY SYSTEM FOR ANTISENSE OLIGONUCLEOTIDES AND ITS IN VITRO INVESTIGATION Antisense oligonucleotides (AS ODNs) are short, single-stranded, synthetic molecules that have complementary sequences of targeted mRNA or pre-mRNA by which the selective hybridization yields the inhibition of gene expression. These molecules are promising molecules in the treatment of diseases such as cancer, AIDS, etc. However, the major problems in using such molecules in the treatment of the diseases mentioned above, are lack of stability and poor penetration into the cell, therefore; a spesific carrier is needed. In this thesis, the complex and nanoparticle forms of AS ODNs were prepared by using a cationic polymer called chitosan. Afterwards, the physicochemical properties such as zeta-potential and particle size of these formulations were measured and the antisense activity of the appropriate formulations were investigated in vitro. The antisense activity of 1:20 and 1:100 (AS ODN: Chitosan) complexes and nanoparticles which have approximately 200 nm particle size, were measured based on the inhibition of Beta-galactosidase coded by pSV-Beta-Gal plasmid followed by the addition of antisense containing formulations into the HeLa cells. Eventually, both forms protected AS ODNs from potential enzymatic degredations. However, due to the strong electrostatic bonds between chitosan and AS ODN in nanoparticle formulations, AS ODN failed to be released and consequently, the antisense activity of nanoparticles were observed to be poor. In 1:100 (-/ +) complex formulations, high antisense activity was observed especially in the case of adding AS ODNs after 24 hours from pDNA transfection into the cell. As a result, chitosan can be considered as a suitable carrier especially for phosphorothioate modified AS ODNs. This study can form the basis for the forthcoming studies related with carrier systems of AS ODNs that will be done with chitosan polymer.