A nanocomplex that is both tumor cell-selective and cancer gene-specific for anaplastic large cell lymphoma
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Many in vitro studies have demonstrated that silencing of cancerous genes by siRNAs is a potential therapeutic approach for blocking tumor growth. However, siRNAs are not cell type-selective, cannot specifically target tumor cells, and therefore have limited in vivo application for siRNA-mediated gene therapy.
In this study, we tested a functional RNA nanocomplex which exclusively targets and affects human anaplastic large cell lymphoma (ALCL) by taking advantage of the abnormal expression of CD30, a unique surface biomarker, and the anaplastic lymphoma kinase (ALK) gene in lymphoma cells. The nanocomplexes were formulated by incorporating both ALK siRNA and a RNA-based CD30 aptamer probe onto nano-sized polyethyleneimine-citrate carriers. To minimize potential cytotoxicity, the individual components of the nanocomplexes were used at sub-cytotoxic concentrations. Dynamic light scattering showed that formed nanocomplexes were ~140 nm in diameter and remained stable for more than 24 hours in culture medium. Cell binding assays revealed that CD30 aptamer probes selectively targeted nanocomplexes to ALCL cells, and confocal fluorescence microscopy confirmed intracellular delivery of the nanocomplex. Cell transfection analysis showed that nanocomplexes silenced genes in an ALCL cell type-selective fashion. Moreover, exposure of ALCL cells to nanocomplexes carrying both ALK siRNAs and CD30 RNA aptamers specifically silenced ALK gene expression, leading to growth arrest and apoptosis.
Taken together, our findings indicate that this functional RNA nanocomplex is both tumor cell type-selective and cancer gene-specific for ALCL cells.
KeywordsJurkat Cell Anaplastic Lymphoma Kinase Anaplastic Large Cell Lymphoma Cell Binding Intracellular Delivery
The discovery of RNA interference (RNAi), the process by which specific mRNAs are targeted for degradation by complementary small interfering RNAs (siRNAs), has enabled the development of methods for the silencing of specific genes at the cellular level [1, 2, 3]. In vitro studies demonstrated that siRNA-mediated silencing of oncogenes induces growth arrest and death of tumor cells, indicating their potential therapeutic value [4, 5, 6, 7]. Although siRNAs are gene specific, they are not cell/tissue-selective and therefore can not specifically target or accumulate in tumor tissues. Therefore, an efficient cell/tissue-specific delivery system is needed to make siRNA-mediated gene therapy a feasible approach. In vivo delivery of functional RNAs can be achieved using either viral carriers or non-viral cationic vectors. Although viral carriers achieve high transfection efficiencies, concerns about their safety, immunogenicity, and latent pathogenic effects have convinced researchers to focus on non-viral cationic carriers [8, 9, 10, 11]. Among these cationic carriers, polyethyleneimine (PEI) has been widely studied due to its high cell transfection efficiency, strong buffering capacity, and ability to release functional nucleic acids from endosomes into the cytoplasm by inducing osmotic endosomal rupture [12, 13, 14, 15, 16, 17, 18, 19]. However, PEI carriers alone are not cell/tissue-type specific, thus reaching tumor sites in vivo requires high treatment dosages of PEI, which may be toxic to normal tissues [20, 21]. This cytotoxicity of PEI has thus far prevented its translation to the clinic . While efforts to synthesize safer PEI analogues are underway, decreasing the required dosage of PEI could also reduce toxicity.
To gain cell specificity, the siRNA delivery system can be combined with a target-specific ligand molecule [23, 24, 25, 26]. Although monoclonal antibodies have been widely used as cell-targeting ligands, mouse monoclonal antibodies are immunogenic in vivo and humanized monoclonal antibodies are very costly and only available for a limited number of ligands. Thus, scientists have searched for other ligand molecules that are easier to produce. Aptamers, short single-stranded oligonucleotides (30-50 bases) represent one such class of new small molecule ligands. In contrast to antibodies, aptamers are small oligonucleotides that exhibit no or minimal antigenicity/immunogenicity, so they are more suitable for in vivo use as diagnostic or therapeutic agents [27, 28, 29]. Recently, a RNA aptamer was developed that specifically binds to the CD30 protein in solution . In addition, we have shown that this RNA aptamer selectively binds to intact CD30-expressing lymphoma cells with binding characteristics similar to a CD30-specific antibody .
Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma [32, 33, 34]. ALCL cells exhibit an abnormal expression of the ALK oncogene and unique surface expression of CD30 [35, 36, 37]. The presence of these distinct molecular markers provides the rationale for development of a lymphoma cell-selective and tumor gene-specific therapeutic approach to treat ALCL. Previous studies demonstrated that siRNA-mediated knockdown of ALK gene expression promotes cell death of ALCL cells [38, 39, 40]. Based on these findings, we hypothesized that ALCL-selective delivery of a tumor gene-specific siRNA could be developed by assembling a functional RNA nanocomplex comprised of the CD30-specfic aptamer and an ALK-targeted siRNA within nano-sized PEI polymer carriers.
Formulation of a nanocomplex containing both CD30 aptamer and ALK siRNA
Zeta potential measurements show that the positive charge of PEI-citrate nanocore (+10 ± 0.6 mV) was reversed after incorporation of the siRNA and aptamer (-41 ± 0.9 mV). This was expected because of the negatively-charged nature of both the siRNA and aptamer that have complexed with the PEI-citrate nanocore. Further, the potential of the nanocomplex dropped to -25 ± 0.9 mV in the cell culture medium due to the high ionic strength.
CD30 aptamers mediate selective ALCL cell binding and intracellular delivery of nanocomplexes
To determine whether binding of the CD30 aptamer-mediated to the cell surface induced nanocomplex internalization, cells were incubated with the test nanocomplexes for 4 hours, the nuclei stained with DAPI, and examined by confocal microscopy. As shown in Figure 5D, intracellular delivery of the nanocomplexes was confirmed by observing the overlap of the Cy5-ssDNA reporter (red) with the DAPI-stained cell nuclei (blue). Control experiments using identically treated Jurkat cells showed no cellular binding or intracellular delivery of the nanocomplex.
Nanocomplexes introduce functional siRNAs into ALCL cell
Nanocomplex treatment silences ALK expression and causes growth arrest of ALCL cells
To examine changes in Karpas 299 cell viability, cells were treated with ALK-siRNA nanocomplexes, as described above, and growth kinetics and cell viability were simultaneously measured at 2 and 4 days post-treatment. Cells were treated for 4 days because the cellular NPM-ALK fusion protein has a long half life time, ≥48 hours. As shown in Figure 7C, treating Karpas 299 cells with the nanocomplex significantly inhibited cell growth (P < 0.05). In contrast, the growth kinetics of nanocomplex-treated Jurkat cells was unaffected. To assess apoptosis, Karpas 299 cells were treated with the nanocomplex for 24 hours, as described above, stained with FITC-conjugated Annexin V, and analyzed by flow cytometry. Nanocomplex-treatment significantly increased the percentage of apoptotic cells from a basal level of 2.2% to 14.1% (Figure 7D, P < 0.05).
An ideal in vivo siRNA carrier system will safely transport the 'cargo' to the desired destination, release a functional cargo in a tissue/cell specific manner, and have no off-target or adverse drug effects. In this study, we have developed this type of carrier system by formulating a functional RNA nanocomplex that is both tumor cell type-selective and cancer gene-specific for ALCL. Advantages of these nanocomplexes include: 1) incorporating siRNAs into a nano-sized carrier will increase their physical size and could prevent the rapid elimination of siRNA from the blood circulation in vivo; 2) incorporating CD30 aptamers will enable specific accumulation of the nanocomplexes within tumor sites and eliminate potential off target side effects of the nanocomplex components; and 3) it is possible to incorporate more than one siRNA and/or therapeutic drug into the nanocomplex to generate additive or synergistic repressive effects on tumor cells. The use of specific ligands for cell targeting and reduction of the PEI dose is critical for in vivo feasibility. PEI polymers have been used for cell transfection at concentrations ranging from 5 to 10 μg/ml [15, 16, 17, 18, 19], at which moderate cytotoxicity has been reported [20, 21]. As demonstrated in this study, incorporating the CD30 aptamer allowed us to use a sub-toxic dose of the PEI carrier in the nanocomplex, less than 1/20 of the reported cytotoxic concentration. It is notable that under in vivo conditions, the CD30 aptamer-mediated cell binding will likely result in an accumulation of the nanocomplexes exclusively in lymphoma tumor tissues and increase the local PEI concentration, possibly reaching a toxic dose. Interestingly, the increased PEI concentration within tumor tissues may enhance the in vivo therapeutic effect of the nanocomplex, but have no adverse effect on normal tissues.
In this study, we have described an approach for developing therapeutic agent by formulating a nanocomplex that is both tumor cell-selective and cancer gene-specific for ALCL. The nanocomplexes are specific and non-cytotoxic to lymphoma cells, which advance great potential for their clinical applications.
Chemical reagents and oligonucleotide synthesis
Branched polyethyleneimine (60 kDa) was purchased from Sigma-Aldrich (Catalog #P3143, St. Louis, MO). Sodium citrate was obtained from Fisher Scientific (Pittsburgh, PA). For silencing the enhanced green fluorescent protein gene (eGFP), eGFP-targeted siRNA was purchased along with a paired control siRNA from Ambion (catalog # AM4626, Foster City, CA). The ALK-targeted siRNA was synthesized by Ambion using the reported sequences: sense, 5'-CACUUAGUAGUGUACCGCCtt-3' and antisense, 5'-GGCGGUACACUACUAAGUGtt-3' . A reporter for the cell binding assays was constructed by synthesizing a single-stranded DNA (ssDNA) oligonucleotide containing the sense ALK siRNA sequence conjugated at the 5' end to the fluorochrome Cy5 (excitation 645 nm/emission 665). The CD30 aptamer was synthesized by Bio-Synthesis (Lewisville, TX), as previously described  using the following sequence: 5'-GAUUCGUAUGGGUGGGAUCGG GAAGGGCUACGAACACCG-3'.
Formulation and characterization of the nanocomplex
To generate the PEI polymer carrier, we used sodium citrate to crosslink PEI molecules. The PEI-citrate core structure (nanocore) was formed by mixing one part by volume of a 100 μg/ml pH 6.0 PEI solution with six parts by volume of sodium citrate. To obtain PEI-citrate nanocores of the optimal size, different 'R' ratios (defined as the ratio of the number of carboxylate groups from citrate to the number of primary amine groups from PEI) were tested. These ratios ranged from 10 to 1 and were obtained by changing the concentration of the citrate solution. The size of the PEI-citrate nanocores produced for each R ratio was determined by obtaining dynamic light scattering measurement (DLS) using a Brookhaven ZetaPALS with a BI-9000AT digital autocorrelator at a wavelength of 656 nm. Diameters were obtained by fitting DLS correlation with the CONTIN routine available through the instrument software 9KDLSW. Electrophoretic mobility was also determined with ZetaPALS using a dip-in (Uzgiris type) electrode in 4-mL polystyrene cuvettes, and the zeta potential was calculated using the Smoluchowski model. To assemble the nanocomplex, three parts by volume of synthetic CD30 aptamers (10nM) and siRNAs (100nM) (or Cy5-labeled ssDNA for validation purposes) were added to the nanocore reaction 5 minutes after initiation and were incorporated into the PEI-citrate nanocores through non-covalent charge forces (Figure 1A). To confirm the colloidal stability of the assembled nanocomplexes, they were incubated in RPMI 1640 cell culture medium at room temperature and the nanocomplex size was monitored by DLS over time.
Cell binding assays
Karpas 299 cells (a human CD30-expressing ALCL cell line from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig) and Jurkat cells (a CD30-negative human leukemia/lymphoma cell line from ATCC, Manassas, VA) were used in this study. Cells (2 × 105) were incubated with PEI-citrate, PEI-citrate/ssDNA(Cy5), or PEI-citrate/ssDNA(Cy5)/Aptamer, as indicated in Figure 5, in 0.5 ml of culture medium for 30 minutes at room temperature. Cell binding of the nanocomplexes was analyzed by flow cytometry (LSRII, BD Biosciences) and fluorescent microscopy (Olympus IX71 inverted microscope) to detect cell surface Cy5 signal. To test their biostability, nanocomplexes were incubated in RPMI 1640 medium, and CD30 aptamer-mediated cell binding was examined at the indicated intervals over 24 hours.
In vitro functional assays
Cytotoxicity assay: the individual components were added into Karpas 299 cell cultures (2 × 105/sample) at their maximal concentrations: 100nM CD30 aptamer, 100 nM ALK gene-targeting siRNA, and 4.2 μM sodium citrate (pH 6.0). After 48 hours, cells were harvested, and cytotoxicity was evaluated by flow cytometry using forward and side scatter parameters. PEI toxicity was also examined by adding serial dilution ranging from 5.48 μg/ml to 0.027 μg/ml to the cell cultures and evaluated as described above. Cell viability assay: cells (2 × 105/sample) were treated as indicated and stained with 0.1% trypan blue in PBS for 15 minutes. Viable cells were counted using a hemocytometer and light microscope. The relative rate of cell growth was determined by calculating the ratio of the number of viable cells in treated samples to the number of cells in the control samples (no treatment). Cell apoptosis assay: cells (2 × 105/sample) were treated for 24 hours as indicated and stained with FITC-conjugated Annexin V using a kit from BD Biosciences. Apoptotic cells were detected by flow cytometry.
Confocal fluorescence microscopy
To demonstrate cell-selective intracellular delivery of the nanocomplex, cultured cells (2 × 105/sample) were treated with the nanocomplex diluted in 0.5 ml of RPMI 1640 medium without serum for 4 hours at 37°C. Cells were then washed twice with PBS and stained with 1 μg/ml 4'-6-diamidino-2-phenylindole (DAPI, Invitrogen) for 15 minutes to label nuclei. Lastly, cells were cytospined onto slide and examined using a laser scanning confocal microscope (Olympus, Fluo ViewTM 1000) at 400 × magnification.
Gene silencing studies
To validate ALCL-selective gene silencing, an eGFP-specific nanocomplex was generated as indicated in Figure 1B and incubated with 2 × 105 eGFP-expressing Karpas 299 or Jurkat cells (40) at PEI concentration of 0.274 μg/ml in 0.5 ml of RPMI 1640 medium for 4 hours at 37°C with or without fetal calf serum as indicated in the figures. The cells were then washed twice with PBS and cultured in medium containing 10% FBS. Expression of eGFP was evaluated by flow cytometry on day 2 post-treatment. Nanocomplexes containing luciferase-targeted siRNAs (Ambion, Austin, TX) were also tested in the luciferase-transfected Karpas 299 or Jurkat cells (40). Changes in luciferase activity in the cultures were evaluated by bioluminescence scanning.
To silence ALK expression, cultured Karpas 299 cells were treated with nanocomplexes containing ALK-targeted siRNAs at a PEI concentration of 0.274 μg/ml, as described above. eGFP-specific nanocomplexes were utilized as an irrelevant gene silencing control. For immunocytochemical studies, cells were harvested at day 2 post-treatment, and cytospins were prepared with a cell preparation kit (BD Biosciences). The cellular nucelophosmin-ALK fusion protein was detected using a mouse anti-human ALK antibody (1:300 dilution, BD Biosciences) and visualized with the DAKO ChemMate detection kit using a horseradish peroxidase-conjugated rabbit anti-mouse antibody and the color development substrate DAB. Images were taken using a light microscope. In addition, ALK fusion protein expression in the treated cells was examined by immunoblotting, as previously described .
All experiments were performed greater than or equal to three times. Data were analyzed by Student's t test. P values of less than 0.05 were considered significant.
This work was supported by the National Institutes of Health/National Cancer Institute grants CA113493 and CA151955 (to Y.Z.) and the Sid W. Richardson Foundation for the Rice University Institute of Biosciences and Bioengineering Medical Innovations Award Grant (to M.S.W.).
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