RESUMO
A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of 33P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Oligonucleotídeos Antissenso/análise , Cianoacrilatos/química , Embucrilato , Marcação por Isótopo , Oligonucleotídeos Antissenso/isolamento & purificação , Radioisótopos de Fósforo , Polímeros/química , Reprodutibilidade dos Testes , Contagem de CintilaçãoRESUMO
PURPOSE: The goal of this study was to evaluate the ability of nanoparticles to be used as a targeted delivery system for oligonucleotides. METHODS: Pharmacokinetic and tissue distribution were carried out in mice by measuring radioactivity associated to the model oligothymidylate 33P-pdT16 loaded to poly(isobutylcyanoacryate) (PIBCA) nanoparticles. In addition, we have used a TLC linear analyzer to measure quantitatively on a polyacrylamide gel electrophoresis, the amount of non degraded pdT16. RESULTS: Organ distribution study has shown that nanoparticles deliver 33P-pdT16 specifically to the liver reducing its distribution in the kidney and in the bone marrow. Nanoparticles could partially protect pdT16 against degradation in the plasma and in the liver 5 min after administration, whereas free oligonucleotide was totally degraded at the same time. CONCLUSIONS: Nanoparticles protect oligonucleotides in vivo against degradation and deliver them to the liver.