RNA aptamer-conjugated liposome as an efficient anticancer drug delivery vehicle targeting cancer cells in vivo
Graphical abstract
Introduction
Targeted delivery of anticancer therapeutic agents such as doxorubicin (Dox) and docetaxel to cancer cells is a highly desirable strategy for the treatment of cancerous tumors without affecting normal cells. To this end, methods for the conjugation of drug delivery materials with various biomolecules and ligands that are specific to cancer cells have been developed and applied for the targeted delivery of anticancer drugs to cancer cells [1], [2], [3]. Surface modification of nanosized carriers such as liposomes, micelles, and polymeric nanoparticles with cancer cell-specific antibodies (Abs) is a common way of identifying cancer cell-surface targets [4], [5], [6], which then uptake the Abs-conjugated drug delivery vehicles via receptor-mediated endocytosis [7], [8]. Although antibody-based drug delivery provides selective and improved therapeutic efficacy against several cancer cells, Abs-conjugations are difficult to control and often have inconsistent binding affinities [9]. In addition, the Abs used as cell-specific homing agents must be optimized for use in humans to be eligible for clinical application [9], [10].
Nucleic acid aptamers have emerged as an alternative to Abs for producing surface-modified nanomaterials for targeted delivery application [11]. Aptamers are single-stranded oligonucleotides that are created using an in vitro selection process termed Systematic Evolution of Ligands by EXponential enrichment (SELEX) [12], [13]. As a nucleic acid analog of Abs, an aptamer can specifically bind to a broad range of targets such as small organic molecules, proteins, viruses, or cells with high affinity. Nucleic acid aptamers have several advantages over protein Abs. Aptamers can be obtained in large amounts through chemical synthesis and are much more resistant to heat, pH change, and organic solvents than Abs [14]. Aptamers can be denatured and renatured without loss of activity and are thought to be less immunogenic than protein Abs due to their gradual degradation by nucleases in vivo. Moreover, aptamers can be chemically modified with diverse functional groups on either the 5′ or 3′ end of the nucleic acid to facilitate site-specific conjugation. Thus, DNA or RNA aptamers have been used as tumor targeting agents in several nanoparticles for targeted drug delivery [9], [15], [16], [17], [18], [19]. Although nanotechnology platforms based on nucleic acids and aptamers are promising approaches for targeted cancer therapy, the clinical efficacy of aptamer-conjugated drug delivery systems for targeted cancer therapy has yet to be demonstrated in vivo.
Of the nanomaterial-based drug delivery systems used for cancer therapy, the liposome-based system is one of the most established and successful platform technologies [20]. Several liposome-based systems have been approved for clinical use by the US Food and Drug Administration [21]. Liposomes are capable of encapsulating highly toxic or poorly soluble pharmaceuticals, and the liposome surface can be modified with polyethylene glycol (PEG) for extended systemic circulation and preferential accumulation at tumor sites [22]. Liposomes can be modified by conjugating nucleic acid aptamers so that the plasma residence time of aptamers increases [23]. Liposome-based drug delivery mainly utilizes a passive targeting mechanism through the so-called enhanced permeation and retention (EPR) effect in cancerous tumor tissues [24], [25]. Thus, functionalization of liposomes with cancer cell-targeting aptamers could, in principle, improve the ability of liposomes to transport drugs with better efficacy and fewer systemic side effects, which would be better than using a liposomal system entirely based on EPR for cancer targeting [5].
Herein, we designed and synthesized drug-encapsulating liposomes conjugated with an RNA aptamer specific to the prostate specific membrane antigen (PSMA), which is expressed on the surface of prostate cancer cells. The RNA aptamer-conjugated liposome, termed an aptamosome, is composed of several phospholipids, including 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), and cholesterol. The aptamosome complex was prepared by inserting the RNA aptamer-conjugated micelles into a POPC-based liposome. We evaluated specific binding and subsequent uptake of the aptamosomes to LNCaP prostate epithelial cells that express PSMA. We next encapsulated the anticancer drug Dox into the aptamosomes and investigated drug efficacy, which is expected to be specific to LNCaP prostate cancer cells. We then used LNCaP xenograft nude mice to assess the biodistribution of Dox-encapsulating aptamosomes and the anticancer efficacy of Dox, which was delivered with the PSMA-specific aptamosomes in vivo.
Section snippets
Materials
POPC, DSPE-PEG2000, DSPE-PEG2000-Mal, DOPE, Rho-DOPE [rhodamine-(1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine)], and cholesterol were purchased from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). Polycarbonate membranes (19 mm in diameter) were purchased from Whatman (Kent, Maidstone, UK). Agarose beads (Sepharose CL-4B) and Dox were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). WST-1 reagent for cell viability assay was purchased from Daeil Lab Service (Seoul, Korea).
Preparation and physical characteristics of PSMA aptamosome
Prostate cancer cells can be targeted with a PSMA-specific RNA aptamer named xPSM-A9 [26], which has been used as a homing agent for delivery vehicles [27]. As shown in Fig. 1A, we synthesized 94 nts RNA molecules that contained the xPSM-A9 sequence (70 nts) and a 3′-end spacer sequence (24 nts) and annealed it to 26 nts linker DNA modified with FITC and thiol at the 5′- and 3′-ends, respectively (Fig. 1A). We then used thiol–maleimide chemistry to conjugate the thiol-modified linker DNA to
Conclusions
We have developed and synthesized a nanosized liposomal material that is conjugated to a PSMA-specific RNA aptamer. Our RNA aptamer-conjugated liposomes specifically bind to the PSMA-positive prostate cancer cells as verified by confocal microscopic images and flow cytometry. Anticancer drug (doxorubicin, Dox) was successfully encapsulated into the RNA aptamer-conjugated liposomes, and the Dox-encapsulating aptamosomes were significantly more toxic to the targeted prostate cancer cells than to
Acknowledgments
This work was supported by the National Research Foundation grants funded by the Korean Government MSIP (NRF-2011-0013997, 2010-0019306 and 2012M3A9B2028336). This paper was written as part of Konkuk University's research support program for its faculty on sabbatical leave in 2014.
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These authors contributed equally to this work.