Design and synthesis of chitosan nanoparticles conjugated with folic acid for targeted transfer of miR-126 to lung cancer cell lines

Design and synthesis of chitosan nanoparticles conjugated with folic acid for targeted transfer of miR-126 to lung cancer cell lines
Fooroogh Nikchehreh Golafzani,¹ Sepideh Khaleghi,^2,* Masoud Javanmardi,³
1. department of medical biotechnology, faculty of advanced science and technology, Tehran medical science, Islamic Azad University, Tehran, Iran.
2. department of medical biotechnology, faculty of advanced science and technology, Tehran medical science, Islamic Azad University, Tehran, Iran.
3. Department of medical biotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Introduction: Introduction: Lung cancer is still the most common cancer globally. Early screening remains the key to improve the prognosis of patients in recent years, studies have found that miRNAs play an important role in lung cancer. MicroRNA-126 is a tumor suppressor microRNA which is significantly down regulated in lung cancer cell lines, such as A549 cells and has been reported to be used as a therapeutic target for lung cancer. However, the delivery of miRNA has always been limited by many barriers. In the present study, miR126 loaded gene delivery system based on chitosan nanoparticles conjugated folic acid was prepared in order to deliver cs-FA-miR126 into A549 cells
Methods: Materials and methods: The nanoparticles were characterized using dynamic light scattering(DLS), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The manner of miRNA loading was speculated by gel retardation assay. Dose dependent cellular toxicity by Cs-FA-mir126 and Cs-FA-miR scramble was demonstrated by MTT. The expression of key genes in apoptosis (BAX, caspase 9) and autophagy (Becline1, ATG5) process were assumed by Quantitative Real Time PCR. The function of nanoparticles in the Apoptosis and cell cycle arresting were evaluated by flow cytometery. Moreover, in vitro nanoparticle efficacy on metastasis inhibiting was evaluated through scratch test.
Results: Results : Nanoparticles were produced based on modified ionic gelation of tripolyphosphate(TPP). DLS confirmed small sized nanoparticles (<200). while FTIR confirmed different functional groups associated with synthesized nanoparticles.MTT analysis results showed that cell viability in cs-FA-mir 126 treatmentation group was significantly lower than that control groups.the result showed that cs-FA-miR126 group overexpression inhibits the A549 cell viability. Flowcytometry results demonstrated that the number of apoptosis cells in cs-FA-miR126 treatmentation groups was significantly higher than that the cs-FA-miR scramble and control groups. the expression of EGFL7 was reduced and the expression of BAX, caspase9, ATG5, Beclin1 were increase remarkably. Cs-FA-miR126 can induce apoptosis of the A549 cells by regulatory the expression of BAX, caspase 9, ATG5, Beclin1.
Conclusion: In conclusion, our study suggest that folic acid conjugated chitosan nanoparticles can mediate the delivery of Mir-126 directly into lung tumor cells while preserving its molecular and biological activity.
Keywords: Key words: Chitosan nanoparticle, miRNA-126, Folic acid, Apoptosis, Autophagy.