Target-specific gene silencing by siRNA plasmid DNA complexed with folate-modified poly(ethylenimine)

https://doi.org/10.1016/j.jconrel.2005.02.006Get rights and content

Abstract

A target-specific delivery system of green fluorescent protein (GFP) small interfering RNA (siRNA) plasmid DNA was developed by using folate-modified cationic polyethylenimine (PEI). A GFP siRNA plasmid vector (pSUPER-siGFP), which inhibits the synthesis of GFP, was constructed and used for suppressing GFP expression in folate receptor over-expressing cells (KB cells) in a target-specific manner. A PEI–poly(ethylene glycol)–folate (PEI–PEG–FOL) conjugate was synthesized as a pSUPER-siGFP plasmid gene carrier. KB cells expressing GFP were treated with various formulations of pSUPER-siGFP/PEI–PEG–FOL complexes to inhibit expression of GFP. The formulated complexes were characterized under various conditions. Their GFP gene inhibition and cellular uptake behaviors were explored by confocal microscopy and flow cytometry analysis. pSUPER-siGFP/PEI–PEG–FOL complexes inhibited GFP expression of KB cells more effectively than pSUPER-siGFP/PEI complexes with no folate moieties and showed far reduced extent of inhibition for folate receptor deficient cells (A549 cells). The results indicated that folate receptor-mediated endocytosis was a major pathway in the process of cellular uptake, suggesting that targeted delivery of siRNA vector could be achieved to a specific cell.

Introduction

RNA interference (RNAi) using small interfering RNA (siRNA, a double-stranded RNA molecule having 21–23 bp) has recently provided a powerful tool for silencing a target gene in gene therapy. RNAi induces highly sequence-specific degradation of homologous mRNA by double-stranded RNA (dsRNA). The process of RNAi is very useful for genetic analysis and is likely to become a potent therapeutic approach for gene silencing [1], [2]. The general mechanism of RNAi involves the cleavage of long dsRNA molecules into 21–23 nucleotides small interfering RNAs (siRNAs) by Dicer, an endogenous RNaseIII-like enzyme [3]. The siRNA is incorporated into a ribonuclear protein complex known as the RNA-induced silencing complex (RISC), which contains the proteins necessary for unwinding the double-stranded siRNA, binding, and cleaving the target messenger RNA [4]. In mammalian cells, exposure to dsRNAs with more than 30 bp in length triggers a sequence-nonspecific interferon response that leads to global inhibition of mRNA translation [5], [6]. However, introduction of shorter siRNAs into mammalian cells results in mRNA degradation with great sequence specificity without the activation of an interferon response [1]. RNAi by synthetic siRNAs of 21–23 nucleotides depresses endogenous and exogenous gene expression in mammalian cells in vitro [7].

Recently, several groups have used siRNA for treating infectious diseases and cancers mediated by variant gene expression [8], [9]. For specific gene silencing in a target tissue, a delivery system of siRNA is highly demanded. In the past decade, various kinds of cell-targeting ligands including antibodies, growth factors, peptides, transferrin, and folate have been conjugated to several types of gene carriers, such as polymer conjugates, liposomes, polymer micelles, and nano-particles for target-specific delivery [10], [11], [12], [13], [14]. Many bioactive agents including plasmid DNA, anti-sense oligonucleotides, anti-cancer agents, and imaging agents could be delivered site-specifically to target cells and tissues [11]. Among them, folate has been popularly used as a targeting ligand for plasmid DNA, doxorubicin, and anti-sense ODN [15].

In this study, an anti-green fluorescent protein (GFP) siRNA plasmid system (pSUPER-siGFP) was constructed and used to inhibit the expression of exogenous GFP in mammalian cells in a target-specific manner. The anti-GFP siRNA plasmid was complexed with a PEI-based cationic polymer conjugate, poly(ethylenimine)–poly(ethylene glycol)–folate (PEI–PEG–FOL), and the complexes were transfected to folate receptor over-expressing cells that produce exogenous GFP. The pSUPER-siGFP/PEI–PEG–FOL complexes were characterized with dynamic light scattering and gel electrophoresis and their cell-specific gene silencing effect was comparatively and quantitatively examined using folate receptor positive cells (KB cells) and folate receptor negative cells (A549 cells). The extent of GFP inhibition in KB cells was also analyzed by flow cytometry.

Section snippets

Materials

Forward and reverse oligonucleotides for cloning anti-GFP siRNA sequence were synthesized and purified by Bioneer (Daejeon, Republic of Korea). A mammalian siRNA expression vector, pSUPER-RNAi (3176 bp), was purchased from Oligoengine (Seattle, WA). Poly(ethylenimine) (branched PEI, MW 25,000) was supplied from Aldrich (Milwaukee, WI). Poly(ethylene glycol) (COOH–PEG–NH2, MW 3400) was obtained from Nektar (Huntsville, AL). Folate and N-hydroxylsuccinimide (NHS) were obtained from Sigma (St.

Results and discussion

Instead of synthetic siRNA, GFP siRNA-expression vector system was constructed and used for GFP gene silencing in a cancer cell. Although direct delivery of synthetic siRNA into the cytosol would be more effective for non-dividing cells, the gene silencing effect is transient and non-inducible [19]. In contrast, the siRNA plasmid vector system requiring intranuclear localization can stably express siRNA in an inducible manner for fast dividing mammalian cells. Thus, for rapidly growing cancer

Acknowledgements

This work was supported by the grant (M1-0214-00-0117) from the Ministry of Science and Technology, Korea.

References (32)

Cited by (118)

  • Targeting nucleic acid-based therapeutics to tumors: Challenges and strategies for polyplexes

    2022, Journal of Controlled Release
    Citation Excerpt :

    The high affinity of folic acid to its receptor has been employed to enhance the efficacy of cancer therapeutics [309,310]. Conjugated to polyplexes, folic acid has achieved enhanced accumulation of both RNAi (siRNA, antisense oligonucleotides and shRNA) [108,274,278–285] and DNA [275–277] therapeutics in tumor cells. Early FR-targeted PLL-based DNA polyplexes exhibited a 6-fold higher gene expression than polyplexes without folate [275].

View all citing articles on Scopus
View full text