Trilysinoyl oleylamide-based cationic liposomes for systemic co-delivery of siRNA and an anticancer drug

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Abstract

Oligolysine-based cationic lipid derivatives were synthesized for delivery of siRNA, and formulated into cationic liposomes. Among various oligolysine-based lipid derivatives differing in lysine residue number and lipid moiety, trilysinoyl oleylamide (TLO)-based liposomes (TLOL) showed the highest delivery efficiency combined with minimal cytotoxicity. Delivery of siRNA using TLOL silenced target genes both in vitro and in vivo. In green fluorescent protein (GFP)-expressing tumor tissue, a significant reduction of fluorescence was observed after intratumoral administration of siGFP using TLOL compared with control siGL2. Intravenous administration of siMcl1 employing pegylated TLOL (pTLOL) reduced the expression of human Mcl1 protein in KB-xenografted tumor tissue. Despite the reduction in target protein Mcl1 expression following such systemic delivery, tumor growth was only slightly reduced compared to a siGL2-treated control group. To potentiate the anticancer activity of siMcl1, the anticancer drug suberoylanilide hydroxamic acid (SAHA) was additionally encapsulated in pTLOL. After intravenous administration of siMcl1 using SAHA-loaded pTLOL (pSTLOL), a significant reduction in tumor growth was observed compared to that seen in animals treated with free SAHA or siGL2 complexed with pSTLOL. The results indicate that pTLOL could be further developed as a systemic delivery system for synergistic anticancer siRNA and a drug.

Graphical Abstract

Intravenous co-administration of siMcl1 and SAHA using pSTLOL could significantly inhibit the growth of tumor tissues.

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Introduction

Although small interfering RNAs (siRNAs) are emerging as a new class of therapeutics, the development of effective delivery systems is critical if such molecules are to be useful as pharmaceuticals in various applications [1], [2]. Several siRNA clinical trials are in progress, treating localized ocular and lung disease [3]. A prime application of new siRNA therapy is anticancer therapeutics [4], [5]. To develop novel anticancer siRNAs, pharmacologically adequate amounts of siRNA must be delivered to tumor tissues, allowing knockdown of target genes/proteins.

Currently, anticancer siRNA delivery systems are under intense investigation [6]. Among various systems studied, cationic lipid-based systems have received a great deal of attention in efforts to provide the requisite silencing activity after local or systemic administration [7], [8]. Amino acids [9], synthetic chemicals [10], and peptides [11] have been conjugated to lipids to synthesize cationic molecules for siRNA delivery. Recently, combinatorial synthesis and screening of cationic lipids have been undertaken to develop new materials with improved delivery efficiency [12]. Although a great deal of progress has been made in this area, more effective siRNA delivery systems are required to reduce the expression of target genes after intravenous administration.

In the present study, we synthesized several oligolysine-based cationic lipid derivatives, and explored whether these materials could systemically silence target genes. Here, we report that trilysinoyl oleylamide (TLO)-based liposomal formulations reduced target gene expression after systemic administration. Moreover, we found that combined delivery of anticancer siRNA and an anticancer drug yielded synergistic anticancer activity.

Section snippets

Materials

Dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DSPE) were purchased from Avanti Polar Lipids (Birmingham, AL). Suberoylanilide hydroxamic acid (SAHA) was from Cayman Chemical (Ann Arbor, MI). Cholesterol (Chol) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, MO).

Preparation of cationic oligolysinoyl lipid-based liposomes

To prepare multilamellar liposomes, cationic oligolysinoyl lipids, DOPE, and Chol

Synthesis of cationic lipids

The synthesis schemes for the various cationic lipids are summarized in Suppl. Figs. 1 and 2. Oligolysinoyl oleylamides (OLO, Fig. 1A) were synthesized from oleylamine and various oligolysines with 1–10 lysine residues. Oligolysine peptides were covalently linked to oleylamine via formation of amide bonds between the amine groups (–NH2) of oleylamine and the carboxyl group (–COOH) of an oligolysine peptide (Suppl. Fig. 1). To synthesize trilysinoyl cationic lipids, the carboxyl group of a

Discussion

In the present study, we have shown that TLO-based cationic liposomes serve as an effective carrier for delivery of siRNA both in vitro and in vivo. Moreover, a combination of siRNA with the anticancer drug SAHA showed enhanced anticancer activity.

DOPE was used as the basic lipid component in the formulation of OLOL. DOPE is a fusogenic lipid, preferentially forming the inverted hexagonal phase typical of membrane fusion events [14]. The presence of DOPE in cationic liposomes enhances cellular

Acknowledgments

This work was supported by research grants from the Ministry of Education, Science and Technology (2010K-001245; 2010K-001356), from the Korean Health Technology R&D project, Ministry for Health, Welfare and Family Affairs (Grant No. A092010), and from the Bio-Green 21 program (Code No. 20100301-061-200-001-03-00), Rural Development Administration, South Korea.

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