1818
Biomacromolecules 2009, 10, 1818–1827
Enhanced Cytoplasmic Delivery of siRNA Using a Stabilized Polyion Complex Based on PEGylated Nanogels with a Cross-Linked Polyamine Structure Atsushi Tamura,† Motoi Oishi,†,‡,§ and Yukio Nagasaki*,†,‡,§,|,⊥ Graduate School of Pure and Applied Sciences, Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), Center for Tsukuba Advanced Research Alliance (TARA), Master’s School of Medical Sciences, Graduate School of Comprehensive Human Science, and International Center for Materials Nanoarchitectonics Satellite (MANA), National Institute for Materials Science (NIMS) and University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8573, Japan Received February 28, 2009; Revised Manuscript Received May 2, 2009
A novel siRNA delivery system using a polyion complex (PIC) based on PEGylated polyamine nanogels composed of a chemically cross-linked poly[2-(N,N-diethylaminoethyl)methacrylate] (PDEAMA) core and surrounded by PEG tethered chains is described. The nanogel formed PIC spontaneously through electrostatic interaction upon mixing with siRNA. The nanogel/siRNA complex was characterized by a gel retardation assay, size and ζ-potential measurements, and gene silencing activity using a cultured cell line. The nanogel/siRNA complexes showed higher polyanion exchange tolerability compared with the noncross-linked PEG-b-PDEAMA/siRNA complexes, indicating that the three-dimensionally cross-linked structure of the nanogel enhanced the stability of the PIC. Furthermore, the nanogel/siRNA complex was observed to undergo a remarkable enhancement of the gene silencing activity against the firefly luciferase gene expressed in HuH-7 cells at low N/P ratios (N/P ) 2), whereas the noncross-linked PEG-b-PDEAMA/siRNA complexes showed negligible gene silencing activity. Moreover, confocal fluorescence microscopy revealed an efficient endosomal escape capability for the transportation of siRNAs into the cytoplasm, presumably due to the buffering effect of the PDEAMA core. Therefore, the PIC of siRNA with cross-linked polyamine nanogel is a potentially effective siRNA carrier for the development of in vivo therapeutic applications of siRNA.
Introduction RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing pathway initiated by small interfering RNA (siRNA),1 which is expected to serve as a potential therapeutic agent in the treatment of various intractable diseases including genetic disorders and cancer.2-4 Nevertheless, the potential therapeutic application of siRNA is restricted because of its low permeability through the cellular membranes and low stability against enzymatic degradation. Moreover, systemically administered naked siRNA rapidly excreted from the bloodstream mainly due to the clearance from kidneys because the molecular weight of siRNA (13-15 kDa) is below the threshold of the renal glomeruli (ca. 50 kDa).5 Accordingly, the therapeutic value of siRNA under in vivo conditions is largely dependent on the development of an effective nanocarrier system, which achieves modulated body distribution throughout the systemic administration and accumulation in tumor tissues through enhanced permeability and retention (EPR) effect.6 In this regard, poly(ethylene glycol) (PEG)-modified polycations have been utilized for complexation with siRNA to form nanosized PEGylated polyion complexes (PICs) composed of a segregated PIC core surrounded by PEG palisade layers to * To whom correspondence should be addressed. Phone: +81-29-8535749. Fax: +81-29-853-5749. E-mail:
[email protected]. † Graduate School of Pure and Applied Sciences. ‡ TIMS. § TARA. | Master’s School of Medical Sciences, Graduate School of Comprehensive Human Science. ⊥ MANA and NIMS.
increase biocompatibility and enzymatic tolerability and prolong the blood circulation time.7-10 Nevertheless, even for the PEGylated PIC, no significant difference in the blood circulation time between naked siRNA and the PEGylated PIC has been observed, thus only 10-20% of the injected dose still remains in the circulation after 15 min.11 Thus, PEGylated PICs including siRNA showed insufficient accumulation in tumor tissue. This means that a considerable amount of the injected PEGylated PICs containing siRNA is excreted from kidneys and partially recognized by reticuloendothelial systems (RESs) such as the liver, spleen, and lung.10 It is most likely that the dissociation of the PEGylated PIC occurred below the critical association concentration (CAC) in the bloodstream, because the electrostatic interaction between the PEGylated polycation and the siRNA seems to be weak under extremely dilute and high ionic strength conditions arising from the small number of anionic charges in the siRNA molecule. Additionally, the entrapped siRNAs in the PEGylated PIC are likely to undergo exchange reactions with endogenous polyanions such as albumin, which leads to the release of siRNA into the bloodstream. Meanwhile, even though the highly stable PEGylated PIC accumulates in the tumor tissue, it is necessary to devise a PEGylated PIC that can escape from the endo/lysosomes to avoid the lysosomal degradation,12 where the pH is 1.4-2.4 units lower than the physiological one (pH ) 7.4).13 In this regard, poly(ethylenimine) (PEI)14 and poly(amidoamine) (PAMAM) dendrimer15 are of interest to accomplish endo/ lysosomal escape by taking advantage of their substantially lower value of apparent pKa, this is the so-called “proton sponge effect” or “buffer effect”.16,17 However, the buffer effect of the
10.1021/bm900252d CCC: $40.75 2009 American Chemical Society Published on Web 06/08/2009
Cross-Linked Polyamine Nanogel for siRNA Delivery
Biomacromolecules, Vol. 10, No. 7, 2009
1819
Figure 1. Schematic illustration of the nanogel/siRNA polyion complex.
polycation with low pKa values occurs only when an excess of amino groups with respect to the siRNA phosphate groups (high N/P ratio) is present in the system, namely, the resulting PIC solution is presumably a mixture of PICs and free polycations. Accordingly, systemic applications of the PEGylated PICs prepared at high N/P ratio are limited by the nonspecific disposition of the free polycations in the body, leading to low gene silencing activity in cancer cells and high toxicity in normal tissues. A promising approach to the systemic application of siRNA is the development of a PEGylated PIC with enhanced stability in the bloodstream as well as efficient gene silencing activity at low N/P ratios. Worth noticing in this regard is a new class of nanosized (
