Elsevier

Biomaterials

Volume 33, Issue 34, December 2012, Pages 8893-8905
Biomaterials

A biomimetic nanovector-mediated targeted cholesterol-conjugated siRNA delivery for tumor gene therapy

https://doi.org/10.1016/j.biomaterials.2012.08.057Get rights and content

Abstract

RNA interference holds tremendous potential as a therapeutic approach of malignant tumors. However, safe and efficient nanovectors are extremely lack for systemic delivery of small interfering RNA (siRNA). The study aimed to develop a biomimetic nanovector, reconstituted high density lipoprotein (rHDL), mediating targeted cholesterol-conjugated siRNA (Chol-siRNA) delivery for Pokemon gene silencing therapy. Chol-siRNA-loaded rHDL nanoparticles (rHDL/Chol-siRNA complexes) were prepared using thin-film dispersion method and their characteristics were investigated in detail. RHDL/Chol-siRNA complexes at the optimal volume ratio (lipid: Chol-siRNA) exhibited high Chol-siRNA-loading efficiency (∼99%), desirable nanoparticle size and excellent stability in serum. In addition, by analyzing Chol-siRNA release profile, rHDL/Chol-siRNA complexes displayed sustained-release characteristic and storage stability. Observations from FACS and confocal microscopic analyses revealed that rHDL-mediated carboxyfluorescein tagged Chol-siRNA (FAM-Chol-siRNA) transfection resulted in highly efficient uptake and specific cytoplasmic delivery of FAM-Chol-siRNA into human hepatocellular carcinoma cell line HepG2 via HDL-receptor mediated mechanism. In vitro cytotoxicity, apoptosis and Western-blot analyses revealed significant cellular growth inhibition and decrease of Pokemon and Bcl-2 protein expression in HepG2 cells treated with Chol-siRNA-Pokemon-loaded rHDL nanoparticles (rHDL/Chol-siRNA-Pokemon complexes), respectively. In in vivo studies, the near-infrared (NIR) dye Cy5 labeled Chol-siRNA-loaded rHDL nanoparticles (rHDL/Cy5-Chol-siRNA complexes) obviously accumulated in tumor of nude mice after i.v. administration as compared with Cy5-Chol-siRNA-loaded lipoplexes (Lipos/Cy5-Chol-siRNA complexes). Morover, rHDL/Chol-siRNA-Pokemon complexes demonstrated great tumor growth inhibition and significant decrease of Pokemon and Bcl-2 protein expression in vivo. These results suggested that rHDL should be an ideal non-viral tumor-targeting vector for Chol-siRNA transfer, and rHDL-mediated Chol-siRNA-Pokemon delivery might be a promising new strategy for gene therapy in hepatocellular carcinoma.

Introduction

RNA interference (RNAi) represents a promising gene silencing technology for functional genomics and a potential gene therapeutic strategy for a variety of genetic diseases [1], [2]. Since the discovery about the RNAi mechanism of gene-specific silencing, synthetic small interfering RNA (siRNA) has been applied widely in the research field of gene therapy [3], [4], as it selectively and persistently suppresses the expression of pathogenic proteins through the sequence-specific degradation of mRNA, with almost no interferon response [5]. However, due to the relatively large molecular weight, polyanionic nature and susceptibility to enzymatic degradation of siRNA duplexes, the effective cellular uptake and intracellular delivery of siRNA remains a major challenge for widespread use of RNAi as a therapeutic modality or even as an investigational tool in vivo [6], [7].

A critical requirement for achieving systemic RNAi in vivo is the introduction of “drug-like” properties, such as stability, cellular delivery and tissue bioavailability, into synthetic siRNA. In exploring the potential of synthetic siRNAs to silence endogenous target genes, chemically modified siRNAs, including cholesterol-conjugated, 2′-O-methyl sugar modified and antibody-linked siRNAs, have been found to markedly improve pharmacological activities in vitro and in vivo [8], [9], [10]. Covalent conjugation of cholesterol to siRNA increases the nuclease–resistant activity of free siRNA, facilitates cellular import and elicits RNAi, which results in silencing of endogenous genes in vivo [9], [11]. New formulation strategies have been reported to form siRNA complexes with several cationic vectors such as polymers, liposomes or peptides [6], [12], [13], but the safety profiles of them remain an obstacle for clinical application. To overcome the cytotoxicity of siRNA vectors caused by excess of positive charge, non-specific interaction and aggregation with serum proteins in the blood, more specific methods are needed for systemic delivery of siRNA.

The most challenging issue in successful application of the RNAi strategy for gene therapy to human neoplasms is the choice of an effective and non-cytotoxic vector for delivering siRNA into tumor cells. High density lipoprotein (HDL) is one of the essential components of the lipid transport system, and plays a critical role in the reverse cholesterol transport from peripheral tissues to liver [14]. Mature HDL consists of apolipoproteins, phospholipid monolayer, cholesterol and hydrophobic core, into which highly hydrophobic drugs can be incorporated [15]. The major protein component (∼70%) in HDL is apolipoprotein A-I (apoA-I), a highly α-helical polypeptide (28 kDa), which promotes cholesterol efflux to cells and is important in maintaining cellular cholesterol homeostasis following interaction of HDL with scavenger receptor class B type I (SR-BI) that is primarily expressed in the liver [16], [17] and most malignant cells [18]. Endogenous HDL particles are of complete biodegradation and non-immunogenicity. Moreover, HDL particles could escape from the reticuloendothelial system thereby exhibiting longer residence time in the circulation [19] than other lipoproteins or most drug formulations. The core contents of the circulating HDL particles are selectively taken up into cells through a SR-BI receptor-mediated mechanism [18]. On the basis of attractive features mentioned above, HDL could be an ideal non-viral siRNA vector, which has been confirmed in Christian et al.’s report [5].

Reconstituted HDL (rHDL) is the synthetic form of the circulating human HDL, and they have similar physicochemical properties. The composition of rHDL includes phospholipids (PC), apoA-I, cholesterol and cholesteryl esters [20]. In the past decades, rHDL has been successfully developed as a drug carrier. RHDL/aclacinomycin (ACM) complexes prepared by sonication of PC, ACM and apoA-I from human serum, kept the basic physical and biological binding properties of native HDL and showed a preferential cytotoxicity for human hepatoma SMMC-7721 cells to normal L02 hepatocytes [21]. RHDL/paclitaxel (PTX) complexes prepared with substantially higher PTX content than that in earlier reports, had superior cytotoxicity against several cancer cell lines that over-expressed SR-BI receptors and the half maximal inhibitory concentration (IC50) had been found to be 5–20 times lower than that of the free drug [22]. Therefore, rHDL was considered attractive as a drug carrier for selective delivery of therapeutic payloads.

Another important consideration about the RNAi strategy for tumor treatment involves the gene, itself, targeted for silencing. Thus far, many gene candidates have been studied. Pokemon, a proto-oncogene, has been recently identified to have crucial but versatile functions in embryonic development, cell differentiation, proliferation and tumorigenesis [23], [24]. Moreover, aberrant over-expression of Pokemon has been observed in many human cancers [25], [26], [27], [28], [29], and its expression levels are effective for the prediction of tumorigenic behavior and clinical outcome [23], [24], [29]. The underlying molecular mechanism of carcinogenesis mediated by Pokemon involves the specific repression of two key tumor suppressive pathways, the alterative reading frame (ARF)-multiple murine double minute gene 2 (HDM2)-p53 pathway [30] and the retinoblastoma (Rb)-early-region-2 transcription factor (E2F) pathway [28]. These findings suggest that Pokemon plays a critical role in cellular transformation and that it may be a potent target of the RNAi strategy for cancer treatment.

In this study, rHDL-based system (Scheme 1) was introduced for targeted Chol-siRNA delivery and gene therapy in the cancer cells that over-expressed SR-BI receptors. The complex-formation and Chol-siRNA-loading efficiency studies were performed to optimize the volume ratio (lipid: Chol-siRNA) in the formulation of rHDL/Chol-siRNA complexes. The incubation of Chol-siRNA-loaded lipoplexes (Lipos/Chol-siRNA complexes) with apoA-I was evaluated by the observations based on dynamic light scattering (DLS) measurement and atomic force microscopy (AFM). The serum stability and Chol-siRNA release behavior of rHDL/Chol-siRNA complexes were investigated in detail. FAM-Chol-siRNA was complexed with rHDL to explore the possibility of transfection in human hepatocellular carcinoma HepG2 cells in vitro. The effects of gene therapy were evaluated by cytotoxicity, apoptosis and Western-blot analyses after transfection of rHDL/Chol-siRNA-Pokemon complexes in HepG2 cells in vitro. In vivo biodistribution and targeting efficiency of Cy5 labeled Chol-siRNA-loaded rHDL nanoparticles (rHDL/Cy5-Chol-siRNA complexes) in tumor-bearing nude mice was investigated using a non-invasive NIR optical imaging technique. Finally, the tumor growth inhibition effect and protein expression levels in vivo were evaluated after i.v. administration of rHDL/Chol-siRNA-Pokemon complexes in a xenograft nude mouse model.

Section snippets

Chemicals

Unmodified siRNA (Nontargeted control siRNA, 5′-UUC UCC GAA CGU GUC ACG UTT-3′), Chol-siRNA (Nontargeted control cholesterol-conjugated siRNA, 5′-Chol-UUC UCC GAA CGU GUC ACG UTT-3′), Chol-siRNA-Pokemon (Chol-siRNA targeting the Pokemon gene, 5′-Chol-GCA CTT TAA GGA CGA GGA CTT-3′), and FAM or Cy5 labeled Chol-siRNA were synthesized and purified with HPLC by Shanghai GenePharma Co., Ltd. (Shanghai, China). Apolipoprotein A-I (apoA-I) was a generous gift from Dr. Meiqing Feng (Pharmacy of Fu Dan

Formation and characterization of rHDL/Chol-siRNA complexes

The main obstacle to achieving in vivo gene silencing by RNAi technologies is safe delivery [1], [33]. To date, multiple delivery systems, e.g., cationic lipids [12], cationic polymers [34] and cell penetrating peptides [13] were investigated in vitro and in vivo. However, the positively charged surface characteristics and non-specificity of cationic gene vectors would cause severe cytotoxicity, wide body distribution and aggregation with serum proteins in the body. To alleviate these problems,

Conclusions

In this study, we successfully developed a biomimetic nanovector rHDL for targeted Chol-siRNA delivery and for Pokemon gene silencing therapy. The well-formed rHDL/Chol-siRNA complexes showed high Chol-siRNA-loading efficiency, desirable size distribution, excellent serum and storage stability. The most important advantage of rHDL was that it could provide highly efficient transfection, direct cytosolic delivery and superior tumor targetability, which decreased the expression levels of Pokemon

Acknowledgments

This study was technically supported by the staff from Center of Biomedical Analysis, Department of Pharmaceutics, China Pharmaceutical University, and financially supported by the National Natural Science Foundation of China (No. 81102398), the Natural Science Foundation of Jiangsu Province (No. BK2011624), the Natural Science Fund for Colleges and Universities in Jiangsu Province (No. 10KJB310007), the Fundamental Research Funds for the Central Universities (No. JKP2011007), the Open Project

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