Gene therapy is attractive as a clinical treatment for cancers and genetic disorders. Antisense oligodeoxynucleotides [ASODNs] are single-strand DNA molecules complementary to regions of a target gene that specifically inhibit gene expression by hybridizing the gene's mRNA . Owing to their potential of selective downregulation of gene expression and modulation of gene splicing, ASODNs have attracted attention as promising therapeutic agents in the gene treatment of diseases including cancers [1–3]. Survivin, a member of the inhibitor of apoptosis gene family of proteins, is selectively overexpressed in most human cancers, but not in normal tissues [4–6]. This makes survivin a target not only for cancer diagnosis, but also for the development of novel gene therapeutic agents. ASODNs as novel anticancer agents are an area of heightened interest in the field of survivin inhibition. However, the practical application of ASODNs has faced challenges due to their susceptibility to degradation by cellular nucleases and limited intracellular uptake [7, 8]. Therefore, efficient gene delivery carrier systems need to be developed to address these problems . Various viral and nonviral delivery system carriers have been utilized to shuttle nucleic acids into cells, including cationic modified viruses, cationic lipids, and polymers, but each system has particular limitations , i.e., severe side effects (e.g., immune response and insertional mutagenesis) of viral carriers and cell toxicity of cationic carriers.
In recent years, nanomaterials as new nonviral gene carriers have attracted much attention [10, 11]. Many inorganic materials including gold, carbon nanotubes, graphene oxide, and various inorganic oxide nanoparticles have been intensively studied [9–16]. Halloysites are an economically and abundantly viable clay material that can be mined from deposits . Halloysite Al2Si2O5(OH)4·nH2O is a naturally occurring two-layered aluminosilicate, chemically similar to kaolin, which has a predominantly high-aspect-ratio hollow tubular structure in the submicrometer range and an internal diameter in the nanometer range . As for most natural materials, the size of halloysite nanotubes [HNTs] generally varies from 50 to 70 nm in external diameter, a ca. 15-nm diameter lumen, and 0.5 to 1 μm in length. The neighboring alumina and silica layers create a packing disorder causing them to curve and roll up, forming multilayer tubes. In each HNT, the external surface is composed of siloxane (Si-O-Si) groups, whereas the internal surface consists of a gibbsite-like array of aluminol (Al-OH) groups. Even though much less studied than carbon nanotubes, due to their interesting structure and features such as unique tubular structure, large aspect ratio, cheap and abundant availability, rich functionality, good biocompatibility, and high mechanical strength, HNTs are attractive materials that show great promise in a range of applications as a nanoscale container for the encapsulation of biologically active molecules (e.g., biocides, enzymes, and drugs), as a support for immobilization of catalyst molecules, controlled drug delivery, bioimplants, and for protective coating (e.g., anticorrosion or antimolding) [19–23]. Despite these prospects, however, their utilization as biocarrier for ASODNs delivery has been less investigated so far.
In the present work, we developed a novel HNT-based drug delivery system containing ASODNs as a therapeutic gene for targeting survivin and functionalized HNTs [f-HNTs] as carriers. Herein, in order to facilitate the loading and intracellular tracking of ASODNs, f-HNTs were obtained by surface modification with γ-aminopropyltriethoxysilane [APTES], and fluorescein [FAM] was used to bind to ASODNs as fluorescent labeling. Furthermore, cellular uptake and delivery efficiency of the f-HNT-ASODN composites as well as cellular apoptosis induced by the ASODNs transfected with f-HNTs were investigated through confocal microscopy and flow cytometry. The results indicated that these natural, cheap, and abundantly available clay nanotubes could be used as novel vectors in the promising application of gene therapy.