Article pubs.acs.org/bc
In Vitro and In Vivo Tumor-Targeting siRNA Delivery Using FolatePEG-appended Dendrimer (G4)/α-Cyclodextrin Conjugates Ayumu Ohyama,†,‡ Taishi Higashi,† Keiichi Motoyama,† and Hidetoshi Arima*,†,‡ †
Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences and ‡Program for Leading Graduate Schools “HIGO (Health life science: Interdisciplinary and Glocal Oriented) Program”, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan S Supporting Information *
ABSTRACT: We previously reported that folate-polyethylene glycol (PEG)-appended dendrimer (generation 3)/α-cyclodextrin conjugate (Fol-PαC (G3)) shows folate receptor-α (FR-α)-overexpressing tumor cell-selective in vitro siRNA transfer activity. However, FolPαC (G3)/siRNA complex did not induce a significant in vivo RNAi effect after intravenous administration to tumor-bearing mice, possibly resulting from immediate dissociation of the complex in blood. Herein, to develop the novel siRNA carrier having high blood circulating ability, high in vivo siRNA transfer activity, and high safety profile, we newly prepared Fol-PαCs with higher generation (G4) and evaluated their potential as tumor-targeting siRNA carriers in vitro and in vivo. Fol-PαC (G4, average degree of substitution of α-cyclodextrin (DSC) 2.9, average degree of substitution of folatePEG (DSF) 2)/siRNA complex had the prominent RNAi effect through adequate physicochemical properties, FR-α-mediated endocytosis, efficient endosomal escape, and siRNA delivery to cytoplasm with negligible cytotoxicity. Importantly, Fol-PαC (G4, DSC2.9, DSF2) improved the serum stability, blood circulating ability, and in vivo RNAi effects of siRNA, compared to FolPαC (G3). Furthermore, Fol-PαC (G4, DSC2.9, DSF2) complex with siRNA against Polo-like kinase 1 (siPLK1) suppressed the tumor growth compared to control siRNA complex. These results suggest that Fol-PαC (G4, DSC2.9, DSF2) has the potential as a novel tumor-targeting siRNA carrier in vitro and in vivo.
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negligibly expresses in normal tissues.2,4−6 In addition, as a cancer progresses, the expression level of FR remarkably increases.7 Therefore, FR is one of the potential candidate for not only a promising marker, but also a target protein for cancer therapy. Previously, we prepared α-cyclodextrin (α-CyD)-grafted starburst polyamidoamine (PAMAM) dendrimer conjugate (α-CDE), and demonstrated that α-CDE (generation 2 or 3, G2 or G3) shows higher gene and siRNA transfer activities than dendrimer alone.8−12 In addition, we also reported that folate-polyethylene glycol (PEG)-grafted α-CDE (G3) (FolPαC (G3)) shows FR-α-overexpressing tumor cell-selective in vitro siRNA transfer activity in the luciferase assay system.13 However, Fol-PαC (G3)/siRNA complex did not significantly induce the in vivo RNAi effect after intravenous administration to BALB/c mice bearing Colon-26 tumor cells (FR-α (+)), possibly resulting from immediate dissociation of the complex in the blood. Moreover, treatment efficiency of Fol-PαC/ antitumor siRNA complex to cancer growth has been unclear. To increase the generation of dendrimer is one of the useful approaches for improvement of its in vitro and in vivo siRNA transfer activity, due to the strong interaction with siRNA.14,15
INTRODUCTION Currently, the research on small interfering RNA (siRNA) has been extended due to its potential as a therapeutic macromolecule. However, efficient and safe siRNA delivery systems are required to accomplish the desired RNA interference (RNAi) effect. The siRNA carriers can be classified into viral and nonviral vectors, and the latter have been widely used due to easy preparation of carrier/siRNA complexes, low cytotoxicity, and lack of immunogenicity. However, siRNA transfer activity of nonviral vectors is markedly low, compared to that of the viral vectors. In addition, binding of serum proteins to carrier/siRNA complexes results in attenuation of the RNAi effect because of its restricting cellular uptake, dissociation of siRNA from the complex, and enzymatic degradation of siRNA. In cancer therapy, to achieve the maximum treatment efficacy of the anti-cancer siRNA, the drug delivery technique is awfully important. To give an active targeting ability to siRNA carriers, chemical modification of tumor targeting ligands is well-known. Of various tumor-specific ligands, folic acid (FA)1,2 has emerged as a notable targeting ligand capable of potent interaction with cancer cells expressing the folate receptor (FR) with high affinity (Kd: 10−9−10−10 M).3 FR is engaged with the cell surface through a glycosylphosphatidylinositol (GPI)anchor, and is highly expressed in various tumor cells including malignancies of the brain, ovary, breast, kidney, and lung, and © XXXX American Chemical Society
Received: October 9, 2015 Revised: December 10, 2015
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DOI: 10.1021/acs.bioconjchem.5b00545 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry However, dendrimers having high generation often show high toxicity.16 Actually, we previously reported that plasmid DNA (pDNA) complex with dendrimer (G4) or α-CDE (G4) shows severe cytotoxicity in NIH3T3 cells and RAW264.7 cells, despite pDNA complexes with dendrimers (G2, G3) or αCDEs (G2, G3) showing negligible cytotoxicity at a charge ratio of 100 (carrier/pDNA). Thus, the utility of dendrimers (
