Functionalized halloysite nanotube-based carrier for intracellular delivery of antisense oligonucleotides
© Shi et al; licensee Springer. 2011
Received: 23 September 2011
Accepted: 28 November 2011
Published: 28 November 2011
Halloysites are cheap, abundantly available, and natural with high mechanical strength and biocompatibility. In this paper, a novel halloysite nanotube [HNT]-based gene delivery system was explored for loading and intracellular delivery of antisense oligodeoxynucleotides [ASODNs], in which functionalized HNTs [f-HNTs] were used as carriers and ASODNs as a therapeutic gene for targeting survivin. HNTs were firstly surface-modified with γ-aminopropyltriethoxysilane in order to facilitate further biofunctionalization. The f-HNTs and the assembled f-HNT-ASODN complexes were characterized by transmission electron microscopy [TEM], dynamic light scattering, UV-visible spectroscopy, and fluorescence spectrophotometry. The intracellular uptake and delivery efficiency of the complexes were effectively investigated by TEM, confocal microscopy, and flow cytometry. In vitro cytotoxicity studies of the complexes using MTT assay exhibited a significant enhancement in the cytotoxic capability. The results exhibited that f-HNT complexes could efficiently improve intracellular delivery and enhance antitumor activity of ASODNs by the nanotube carrier and could be used as novel promising vectors for gene therapy applications, which is attributed to their advantages over structures and features including a unique tubular structure, large aspect ratio, natural availability, rich functionality, good biocompatibility, and high mechanical strength.
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.
Materials and methods
All reagents used were available commercially and were of high purity grade. The survivin ASODN sequence used in the current work was 5'-CCCAGCCTTCCAGTCCCTTG and modified with fluorescently labeled on 5' end (FAM-CCCAGCCTTCCAGTCCCTTG-3'), which were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). HNTs were purchased from NaturalNano. Inc. (Rochester, NY, USA). APTES were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Synthesis of f-HNT-ASODN complexes
The f-HNTs and f-HNT-ASODN complexes were characterized with transmission electron microscopy [TEM], dynamic light scattering [DLS] (Malvern Zetasizer NanoZS90, Malvern Instruments, Ltd., Worcestershire, UK), UV-visible [UV-Vis] spectrophotometry (Thermo Multiskan Spectrum, Thermo Scientific, Waltham, MA, USA), and fluorescence spectrophotometry (Varian Cary-Eclipse 500, Varian Medical Systems, Palo Alto, CA, USA).
Cellular uptake of the f-HNT-ASODN complexes
Transmission electron microscopy imaging assay
HeLa cells were seeded at a density of 1 × 106 cells in a 60-mm tissue culture dish and grown overnight. The cells were incubated with the f-HNT-ASODN complexes for 6 h, and then the cells were washed thoroughly with chilled phosphate-buffered saline [PBS], centrifuged into a small pellet, and fixed with 2% glutaraldehyde in PBS (0.01 M, pH 7.4) for 120 min, and then washed three times with PBS (10 min every time). The cells were postfixed with 1% osmium tetroxide in the same buffer for 30 min, then washed three times with PBS, dehydrated through a series of alcohol concentrations (30%, 50%, 70%, 90%, 100%), embedded in Epon, and sliced to a thickness of 70 nm. Images of the sliced images were recorded at 100 kV using a Hitachi 600 TEM microscope (Hitachi High-Tech, Minato-ku, Tokyo, Japan).
Confocal microscopy assay
HeLa cells were seeded at 3 × 104 cells in a 35-mm Petri dish and were cultured in β-methoxyethoxymethyl ether [MEM] containing 10% fetal bovine serum [FBS] at 37°C with 5% CO2. After cell attachment overnight, the HeLa cells were treated with f-HNT-ASODN complexes (1.25 μg/mL), incubated for an additional 4 h in fresh media, and washed by PBS (pH 7.4) three times before confocal imaging. The cellular uptake of the f-HNT-ASODN complexes was examined by confocal laser microscopy (Carl Zeiss LSM 5 PASCAL, Oberkochen, Germany). An argon laser for FAM excitation at 488 nm was used for imaging, and an oil immersion objective (Plan Apo, SEIWA OPTICAL AMERICA INC., Santa Clara, CA, USA; magnification = 63 × 1.4) was used for cellular fluorescence imaging.
Flow cytometry analysis
HeLa cells were seeded in six well plates at a density of 2.5 × 105 cells/well and incubated in MEM cell culture media for 24 h at 37°C and 5% CO2. The cells were then incubated with f-HNT-ASODN-FAM conjugates in MEM cell culture media, and after incubation for 4 h at 37°C and 5% CO2, the cells were detached using trypsin, centrifuged at 1, 000 × g for 10 min, and analyzed using a flow cytometer (SE Diva, BD FACSVantage, Franklin Lakes, NJ, USA). A total of 1 × 105 cells were collected and analyzed for each sample. Three replicates were done for each sample. The untreated cells were used as control. The delivery efficiency was calculated as the percentage of fluorescent cells out of the total number of cells. Fluorescence was detected from the FAM labeled on ASODNs at 488-nm excitation.
In vitro cell toxicity assay of the f-HNT-ASODN complexes
HeLa cells were cultured in a MEM (Gibco, Life Technologies, Invitrogen Co., Carlsbad, CA, USA) medium supplemented with 10% FBS for 12 h at 37°C with 5% CO2. For in vitro cell toxicity assay, cells were seeded into 96 well plates at a density of 1 × 104 cells/plate and treated with ASODNs (150 nM), f-HNTs (1.25 μg/mL), and f-HNT-ASODNs, respectively. After incubation for 24, 48, and 72 h, relative cell viability was measured by standard MTT assay. In this assay, the cell viability was assessed by monitoring the enzymatic reduction of yellow tetrazolium MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) to a purple formazan, as measured at 540 nm (Thermo Multiskan spectrum, Thermo Scientific, Waltham, MA, USA). All experiments were done in six copies and illustrated as average data with error bars.
Results and discussion
Zeta potentials of various samples dispersed in aqueous solution
Zeta potential (mV)
Flow cytometry enabled the quantitative assay of delivery into the cells. HeLa cells incubated with the f-HNT-ASODN complexes for 4 h were analyzed using flow cytometry to evaluate the delivery efficiency of the complex and using FAM as fluorescence labeling. Figure 4c showed the cellular delivery efficiency of f-HNT-ASODNs estimated to be 98.69%, indicating that the f-HNTs had high intracellular delivery ability for ASODNs. Therefore, f-HNTs could be effective in transporting DNA inside the cells and could be utilized as efficient gene delivery vectors, which were mostly attributed that the stable f-HNT complex with high loading capacity could prevent DNA from enzyme degradation
In summary, we have prepared a novel gene delivery system with f-HNTs as carrier for loading and intracellular delivering of ASODNs. The obtained results exhibited that f-HNT-ASODN complexes could efficiently improve intracellular delivery and enhance antitumor activity of ASODNs transfected by the nanotube carrier. Therefore, with the benefits of having a unique tubular structure, large aspect ratio, abundant availability, good biocompatibility, and high mechanical strength, the HNTs could hold a great promise as a viable and inexpensive nanocarrier for biological delivery applications and gene therapy.
This work was supported by the program for New Century Excellent Talents in University (NCET-08-0897), Shanghai Education Committee (09SG43, S30406), National 973 Project (2010CB933901), and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University (DZL806).
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