Naslov
Chitosan-based nanoparticles for siRNA mediated Hsp70 knockdown promote apoptosis in cancer cells

Autori
Mirela Matokanović, Anita Hafner, Jelena Filipović-Grčić, Dusica Maysinger, Karmela Barišić

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of abstracts of the 9th Central European Symposium on Pharmaceutical Technology / - , 2012

Skup
9th Central European symposium on Pharmaceutical Technology

Mjesto i datum
Dubrovnik, Hrvatska, 20.09.2012. - 22.09.2012

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
chitosan; RNA interference; Hsp70; cancer

Sažetak
Introduction: Chitosan is a polymer that has beneficial qualities for in vivo application, such as low toxicity and low immunogenicity. Due to its positive charge, chitosan can easily form polyelectrolyte complexes with negatively charged siRNA by electrostatic interaction, and therefore chitosan-based polymer carriers gained great interest as a delivery system for siRNA [1]. RNA interference (RNAi) is a mechanism for inhibiting gene expression that utilizes endogenously present cellular machinery. The basic principle of RNAi involves the destruction of messenger RNA (mRNA) upon interaction with homologous double stranded RNA (dsRNA), which is also known as small interfering RNA (siRNA). This cellular event leads to downregulation of targeted genes [2]. Apoptosis resistance of cancer cells is associated with increased expression of inducible heat shock proteins (Hsp). siRNAs targeted to Hsp gene family can be introduced into cancer cells by different means of delivery, in order to accomplish greater sensitivity to apoptosis promoted by therapeutics [3]. The aim of this study was to investigate the efficiency of chitosan-based nanoparticles in siRNA delivery (targeted against Hsp70), their silencing efficiency and the capability of inducing apoptosis in cancer cells in vitro. Results and discussion: In our study, solutions of chitosan (in acetate buffer pH 4, 5), in the form of glutamate salt (molecular weight <200kDa ; deacetylation 75-90%), sodium tripolyphosphate and siRNA were used for nanoparticles preparation with chitosan amino groups (N) to RNA phosphate groups (P) ratio of 100:1 [4]. The average particle size and zeta potential were determined and shown to be in the nano-range (170-220 nm). Surface charge of prepared nanoparticles was about +30 mV. Colorimetric MTT assay was performed to show low chitosan-based nanoparticle toxicity in time and dosage manner. Quantitative RT-PCR was used for measuring silencing effect at transcriptional level. Concomitantly, western blot analysis was performed in order to demonstrate the effectiveness of chitosan-based nanoparticles in Hsp70 knockdown at protein level. Celastrol [5] was used as a model chemotherapeutic to study combined effect with Hsp70 gene silencing. Apoptosis accomplished in this manner was measured with a quantitative ELISA for apoptotic cell death. References: [1] Mao, S. et al. (2010) Chitosan-based formulations for delivery of DNA and siRNA. Adv Drug Delivery Rev 62: 12–27 [2] Kong, Y. et al. (2007) RNA interference as a novel and powerful tool in immunopharmacological research. Int Immunopharmacol 7: 417– 426 [3] Ciocca, DR. et al. (2005) Heat shock proteins in cancer: diagnostic, prognostic, predictive and treatment implications. Cell Stress Chaperones 10: 86-103

Izvorni jezik
Engleski

Znanstvena područja
Farmacija

POVEZANOST RADA