Background

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 [1–3]. In vitro studies demonstrated that siRNA-mediated silencing of oncogenes induces growth arrest and death of tumor cells, indicating their potential therapeutic value [

Methods

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' [38]. 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 [31] 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 [43].

Statistical analysis

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.