Enhanced cellular uptake of a triplex-forming oligonucleotide by nanop
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《核酸研究医学期刊》
1 Department of Medicine and 2 Department of Environmental and Occupational Medicine, 3 Environmental and Occupational Health Sciences Institute and 4 The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA
*To whom correspondence should be addressed at UMDNJ-Robert Wood Johnson Medical School, 125 Paterson Street, CAB 7090, New Brunswick, NJ 08903, USA. Tel: +1 732 235 8460; Fax: +1 732 235 8473; Email: thomastj@umdnj.edu
ABSTRACT
We used polypropylenimine dendrimers for delivering a 31 nt triplex-forming oligonucleotide (ODN) in breast, prostate and ovarian cancer cell lines, using 32P-labeled ODN. Dendrimers enhanced the uptake of ODN by 14-fold in MDA-MB-231 breast cancer cells, compared with control ODN uptake. Dendrimers exerted their effect in a concentration- and molecular weight-dependent manner, with generation 4 (G-4) dendrimer having maximum efficacy. A similar increase in ODN uptake was found with MCF-7 and SK-BR-3 (breast), LNCaP (prostate) and SK-OV-3 (ovarian) cancer cells. The dendrimers had no significant effect on cell viability at concentrations at which maximum ODN uptake occurred. Thymidine incorporation showed that complexing the ODN with G-4 significantly increased the growth-inhibitory effect of the ODN. Western blot analysis showed a significant 65% reduction of c-myc protein level in ODN–G-4 treated cells compared with that of ODN-treated/control cells. Gel electrophoretic analysis showed that ODN remained intact in cells even after 48 h of treatment. The hydrodynamic radii of nanoparticles formed from ODN in the presence of the dendrimers were in the range of 130–280 nm, as determined by dynamic laser light scattering. Taken together, our results indicate that polypropylenimine dendrimers might be useful vehicles for delivering therapeutic oligonucleotides in cancer cells.
INTRODUCTION
Gene therapy relies on the efficient transport of oligonucleotides and plasmid DNA through the cell membrane by mechanisms that are not well defined at present (1). Therapeutic oligonucleotides (ODNs) offer the possibility of disrupting the expression of disease-related genes using short synthetic nucleic acid sequences (2–4). Gene expression can be disrupted at the transcriptional (triplex DNA) or translational (antisense DNA) level. In the triplex DNA-based anti-gene approach, transcription is disrupted by the binding of a triplex-forming oligonucleotide at the promoter region of a target gene (5). In contrast, antisense ODN is designed to specifically bind to short complementary sequence of target mRNA in order to prevent the translation of the target gene (6). The ODN is expected to hybridize with the mRNA and block either translational initiation or elongation of transcripts. Because of the high affinity and selectivity of these ODNs for their target sites, they are expected to exert a high level of specificity, and hence represent an interesting class of drugs. Despite this theoretical scenario, there are several limitations in advancing ODN therapeutics to the clinic. For the clinical applications, an important criterion is the availability of a suitable carrier that can efficiently transport and release the ODN at the desired intracellular sites at a controlled rate (7).
There has been much interest in developing new formulations and materials for delivering ODNs to cellular targets in vitro and in vivo. Viral delivery systems have been used in many applications and clinical trials; however, the immune response to viral proteins continues to be a daunting problem (8,9). Lipid-based systems represent a promising class of gene carriers; however, their toxicity on repeated dosing and poor in vivo performance are major drawbacks in advancing them to the clinic (10). There has been considerable interest in recent years in developing polymeric material, such as polyethylenimine (PEI), as gene delivery vehicles (11). PEI is efficient in gene delivery both in vitro and in vivo (12,13). An advantage of PEI is that it allows efficient gene transfer without the need for agents facilitating endosomal escape. Protonated amino groups of PEI appear to buffer acidic endosomal compartment and release DNA to the cytoplasm (14). The transfection efficiency and cytotoxicity of this polymer are dependent on its molecular weight and polydispersity (15,16).
While polymers such as PEI have a range of molecular weight distribution, the novel highly branched three-dimensional molecules, called dendrimers, have defined molecular weight and a large number of controllable peripheral functionalities (17). Tomalia et al. (18) reported the first preparation of an entire series of dendrimers possessing trigonal, 1- > 2 N-based, branching centers. Compared with linear and branched polymers, the monodispersity and controllable surface functionality make the dendrimers an interesting class of gene delivery vehicles. Of the vast majority of dendrimers synthesized, only a few are water soluble and non-toxic, thus rendering them useful to the pharmaceutical industry. Haensler and Sz?ka (19) first showed that polyamidoamine (PAMAM) dendrimers were efficient gene transfer agents for a variety of cultured mammalian cells. Polypropylenimine (PPI) dendrimers, DAB-dendr-(NH2)x, contain terminal amino groups similar to PAMAM. Repetitive addition of a primary amine to two equivalents of acrylonitrile, followed by catalytic hydrogenation, produces polyamine-terminated PPI dendrimers (17,20). A recent report showed that generation 2 and generation 3 PPI dendrimers were effective in transfecting a plasmid DNA in A431 cells (21). In the present study, we investigated the efficacy of five generations of PPI dendrimers to deliver a 31 nt triplex-forming oligonucleotide targeted to the c-myc oncogene (22) in breast, prostate and ovarian cancer cell lines. Our results show that the dendrimers are capable of facilitating the uptake of the ODN in several cancer cell lines in a concentration- and structure-dependent manner.
MATERIALS AND METHODS
Oligonucleotide
HPLC-purified 31 nt phosphodiester ODN was purchased from Oligos, Etc. (Wilsonville, OR). The sequence of the triplex-forming ODN used in our study is: d(GTGGTGG GGTGGTTGGGGTGGGTGGGGTGGG). This sequence is targeted to a triplex-forming region in the promoter of the c-myc oncogene (22,23). As a control, we used d(GGGT GGGGTGGGTGGGGTTGGTGGGGTGGTG), a 31 nt ODN with reversed orientation that could still form G-quartets. The triplex-forming 31 nt ODN was 5'-end labeled with ATP using T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN). Unincorporated label was removed by passing the solution through a Chroma Spin Column (Clontech Lab, Inc., Palo Alto, CA).
Dendrimers, chemicals and other reagents
Dendrimers, DAB Am-4 (polypropylenimine tetraamine dendrimer, generation-1; G-1), DAB Am-8 (polypropylenimine octaamine dendrimer, generation-2; G-2), DAB Am-16 (polypropylenimine hexadecaamine dendrimer, generation-3; G-3), DAB Am-32 (polypropylenimine dotriacontaamine dendrimer, generation-4; G-4) and DAB-Am-64 (polypropylenimine tetrahexacontaamine dendrimer generation-5; G-5) were purchased from Aldrich Chemical Co. (Milwaukee, WI). The chemical structures of the dendrimers are given in Figure 1. Fetal bovine serum (FBS), cell culture media, antibiotics and other chemicals were purchased from Sigma Chemical Co. (St Louis, MO). Monoclonal anti-c-myc antibody and secondary anti-IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used for western blot analysis of c-myc protein levels. Anti-?-actin monoclonal antibody was purchased from Sigma Chemical Co. DNase I was from Roche Diagnostics (Nutley, NJ).
Figure 1. Chemical structures of polypropylenimine dendrimers used in this study. The designations are: G-1, generation 1 dendrimer; G-2, generation 2 dendrimer; G-3, generation 3 dendrimer; G-4, generation 4 dendrimer; G-5, generation 5 dendrimer.
Cell culture
Three breast cancer cell lines (MDA-MB-231, MCF-7 and SK-BR-3), a prostate cancer cell line (LNCaP) and an ovarian cancer cell line (SKOV-3) were obtained from ATCC (Manassas, VA). MDA-MB-231 cells were maintained in minimal essential medium (MEM), supplemented with 10% FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamycin, 0.4 mM sodium pyruvate and 2 mM L-glutamine. MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamycin, 0.4 mM sodium pyruvate, 10 mM non-essential amino acids and 2 mM L-glutamine. Phenol red-free DMEM, containing 10% FBS , was used for experiments using MCF-7 cells to avoid the effects of serum-derived estrogenic compounds and phenol red (24). LNCaP cells were maintained in RPMI-1640 medium containing 10% serum, 10 mM HEPES, 1 mM sodium pyruvate and 2 mM L-glutamine. SK-BR-3 and SKOV-3 cells were grown in McCoy’s 5A medium containing 10% FBS and 3 mM L-glutamine.
Measurement of 32P-labeled ODN uptake
32P-Labeled ODN was added to different concentrations of dendrimer, mixed by vortexing for 5 min, and incubated at 22°C for 1 h. The final concentration of dendrimers ranged from 0.01 to 2.5 μM and the probe was used at 2.5 x 105 c.p.m. (0.4 nM). The complex was added to cells seeded in 6-well plates. Uptake of 32P-labeled ODN was determined as described below. Cells (5 x 105/well) were seeded in 2 ml of medium in 6-well plates and allowed to attach to the plates for 24 h (23). Prior to the experiment, the medium was removed and replaced with 0.5 ml per well of fresh medium, pre-warmed to 37°C. 32P-Labeled ODN (2.5 x 105 c.p.m. level) was added to the medium and the cells incubated at 37°C in a 5% CO2 incubator. The cells were harvested after 4, 6, 8 and 24 h of treatment. The medium was removed, and cells were washed three times with ice-cold phosphate-buffered saline (PBS). A 1 ml aliquot of 1 M NaOH was added to each sample and kept at 60°C for 30 min. The lysate was neutralized with 1 ml of 1 M HCl, and radioactivity quantified using a Beckman (LS 5000 TD) Scintillation Counter. ODN uptake was studied in MDA-MB-231, MCF-7, SK-BR-3, LNCaP and SKOV-3 cell lines.
Cell viability in the presence of dendrimer
Cell viability was determined after adding different concentrations of dendrimers to MDA-MB-231 cells. Cells were plated in 24-well plates at a density of 3.0 x 104 cells/well. Cells were dosed with different concentrations of the dendrimers of different generations. Re-dosing was done every 48 h, after replacing the medium with fresh medium. The number of viable cells was determined every 24 h for 5 days, using trypan blue exclusion assay. Cell viability was calculated as follows:
Cell viability = (no. of viable cells in the sample/no. of viable cells in the control) x 100.
PAGE of 32P-labeled ODN
MDA-MB-231 cells were incubated with 32P-labeled ODN or 32P-labeled ODN–G-4 dendrimer complex at 37°C for 48 h. Cells were harvested at 4, 6, 8, 24 and 48 h after treatment. Cells (in plates) were first washed three times with PBS and then incubated for 2 min with 300 U/ml of DNase I in 50 mM Tris–HCl pH 7.6 containing 10 mM MgCl2. Cells were then washed with PBS, trypsinized, collected in 1 ml of PBS, centrifuged at 1000 g for 5 min, and the supernatant removed. The cell pellet was lysed with 500 μl of 1% SDS lysis buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl and 1% SDS). The lysate was centrifuged at 3000 g for 15 min and supernatant collected in microcentrifuge tubes. ODN was precipitated by adding 50 μl of 5 M NaCl and 1 ml of cold 100% ethanol. After 1 h at –70°C, the solution was centrifuged at 12 000 g for 15 min. The supernatant was removed and the precipitate dried in air. Samples were dissolved in 50 μl of a solution containing 10 mM Tris–HCl and 50 mM NaCl, and analyzed by gel electrophoresis using a 20% polyacrylamide gel at 250 V for 3 h. The gel was dried, and exposed to X-ray film.
Thymidine incorporation assay
MDA-MB-231 cells were seeded in a 6-well plate at a density of 5 x 105 cells/well. After 24 h incubation at 37°C, triplicate wells were treated with ODN alone or ODN–dendrimer complex. ODN was complexed with dendrimer at 100-fold higher concentrations in order to obtain the desired final concentrations in the cell culture medium. DNA synthesis was quantified at 24, 48 and 72 h. For this purpose, 4 μCi/well of thymidine was added 1 h prior to the specified time points. After 1 h incubation at 37°C, cells were washed twice with ice-cold PBS and equilibrated with ice-cold 5% trichloroacetic acid (TCA) for 5 min. It was again washed twice with ice-cold 5% TCA. The cells were then dissolved in 1 M NaOH and neutralized with 1 M HCl. The radioactive thymidine incorporated in the cellular DNA was estimated by liquid scintillation counting.
As a control, the thymidine incorporation assay was repeated with the reversed orientation 31 nt ODN as a single agent or after complex formation with G-4 dendrimer.
Western blot analysis of c-myc protein
The levels of c-myc protein in control and ODN–G-4-treated MDA-MB-231 cells were determined by western blot analysis. MDA-MB-231 cells were seeded in 100 mm dishes at a density of 2 x 106 cells/well. After 24 h incubation at 37°C, cells were treated with ODN, G-4 dendrimer or ODN + G-4 dendrimer in combination. Cells were harvested after 72 h of treatment. Harvested cells were washed with PBS and homogenized by sonicating in lysis buffer containing 150 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 50 mM sodium fluoride, 0.2% SDS, 1 mM sodium vanadate, 2 μg/ml leupeptin, aprotonin and pepstatin, and 1 mM phenylmethylsulfonyl fluoride. A 30 μg aliquot of protein (determined by the Bradford protein assay) was diluted in 2x SDS–PAGE Laemmli buffer (150 mM Tris base pH 6.8, 30% glycerol, 4% SDS, 7.5 mM dithiothreitol, 0.01% bromophenol blue) and separated on a 10% SDS–polyacrylamide gel. After electrophoresis, separated proteins were transferred to PVDF immobilon membrane (Bedford, MA). The membrane was incubated in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, and a 1:200 dilution of monoclonal anti-c-myc antibody overnight. After washing, the membrane was incubated with a 1:2000 dilution of the secondary antibody for 1 h. Blots were then developed by the enhanced chemiluminescence detection technique. To verify equal protein loading, membranes were stripped and re-blotted with anti-? actin monoclonal antibody (1:5000). The intensity of the protein bands was quantified using Kodak 1D Image Analysis Software with a TWAIN Scanner. Lightly exposed films were used for quantification.
Particle size of ODN nanoparticles
The particle size of nanoparticles formed by complexing of ODN with PPI dendrimers was determined using dynamic laser light scattering equipment (25). Experiments were conducted using a DynaPro model MSX (Protein Solutions, Inc., Charlottesville, VA), equipped with a 2 W laser of 800 nm wavelength. Complexation of ODN with dendrimer was done in a buffer containing a physiological concentration of cations (120 mM KCl, 10 mM NaCl, 2 mM MgCl2 and 0.1 mM CaCl2). Then 40 μl of the solution was transferred to the standard quartz cuvette, and the hydrodynamic radii of the particles were determined using dynamic laser light scattering at an angle of 90° at 22°C (25). Data analysis was performed using a Dynamics Version 6 software package supplied by the manufacturer.
RESULTS
Uptake of ODN by MDA-MB-231 cells
Dendrimers of five generations, which differed in molecular weight and number of peripheral amino groups, were used for the uptake study. 32P-Labeled ODN (2.5 x 105 c.p.m./well) was mixed with different concentrations of dendrimers in sterile water. The 32P-labeled ODN–dendrimer complex was added to MDA-MB-231 cells in culture and incubated for 4, 6, 8 or 24 h. The amount of 32P-labeled ODN taken up by cells was quantified by scintillation counting (23). Figure 2 shows the uptake of 32P-labeled ODN after complexing it with G-4 dendrimer. There was a concentration- and/or time-dependent increase in uptake for both control and dendrimer-treated samples. The increase in 32P-labeled ODN uptake was 14-fold higher than that of control, in the presence of 0.1–0.25 μM concentrations of dendrimer at 4 h of treatment, and the enhanced uptake was maintained even after 24 h of treatment. However, there was a decrease in 32P-labeled ODN uptake at the 24 h time point.
Figure 2. Cellular uptake of 32P-labeled ODN by MDA-MB-231 cells, after complexing the ODN with G-4 dendrimer. Cells were treated with 250 000 c.p.m. (0.4 nM) of 32P-labeled ODN after complexing it with G-4 dendrimer in 6-well plates. At the indicated times, the medium was removed, cells washed three times with cold PBS, lysed and cell-associated radioactivity quantified by scintillation counting. Control indicates the use of labeled ODN in the absence of dendrimer. The percentage increase in uptake is calculated with reference to the control group. Results presented are the mean of three separate triplicate measurements. Error bars indicate the SEM.
Figure 3 shows the effects of different generations of dendrimers on 32P-labeled ODN uptake after cells were treated with 32P-labeled ODN complexed with dendrimers. In all cases, there was a concentration-dependent increase in 32P-labeled ODN uptake up to 0.1 μM dendrimer concentration, and then the effect leveled off. G-4 dendrimer was the most efficacious, showing a 14-fold increase in 32P-labeled ODN uptake compared with control. G-5 dendrimer was less efficient than G-4, giving a maximum 8-fold increase compared with control. Table 1 presents the concentrations required for maximal 32P-labeled ODN uptake, using different generations of dendrimers. G-4 dendrimer exerted optimum efficiency at 0.05 μM concentration.
Figure 3. Concentration-dependent increase in the cellular uptake of 32P-labeled ODN in MDA-MB-231 cells in the presence of different dendrimers. Cells were treated for 6 h with labeled ODN–dendrimer complex. The symbols are as follows: G-1 dendrimer, closed circle; G-2 dendrimer, inverted open triangle; G-3 dendrimer, closed square; G-4 dendrimer; open diamond; and G-5 dendrimer, closed triangle. Results presented are the mean of three separate triplicate measurements. Error bars indicate the SEM. In some cases, the error bars are smaller than the size of the symbols.
Table 1. Concentrations of polypropylenimine dendrimers that caused maximum uptake of 32P-labeled ODN in MDA-MB-231 cells
Figure 4 shows the time-dependent increase in 32P-labeled ODN uptake at 0.1 μM concentration of the dendrimers. The efficiency of the dendrimers in facilitating 32P-labeled ODN uptake increased with molecular weight of the dendrimer, except for G-5 dendrimer. Uptake efficiency remained high for G-4 dendrimer at 4, 6 and 8 h, but decreased at the 24 h time point.
Figure 4. Uptake of 32P-labeled ODN in MDA-MB-231 cells after complexing it with a fixed concentration (0.1 μM) of G-1, G-2, G-3, G-4 or G-5 dendrimers. Cells were treated with the indicated dendrimer–ODN complex, and harvested at 4, 6, 8 or 24 h. Results presented are the mean ± SEM.
Uptake of 32P-labeled ODN by other cancer cell lines
In order to examine whether the effectiveness of dendrimers in increasing the uptake of the 31 nt ODN is cell type specific, we next studied the ability of the dendrimers to alter the uptake of the 32P-labeled ODN in SK-BR-3, MCF-7, LNCaP and SKOV-3 cells. Figure 5 shows the uptake of ODN by different cell lines after complexing the ODN with G-4 dendrimer. Although there were cell-specific differences in the maximal uptake of the ODN, the dendrimers enhanced the uptake of the 31 nt ODN in all cell lines tested. There was a 23-fold increase in 32P-labeled ODN uptake compared with control in the ovarian cancer cell line, SK-OV-3, at the 4 h time point in the presence of 0.5 μM dendrimer. However, the effectiveness of the dendrimer on the uptake and/or retention of 32P-labeled ODN decreased with time, demonstrating only a 6-fold increase at the 24 h time point, compared with the control. In SK-BR-3 breast cancer cells, there was a 19-fold increase in the uptake of the 32P-labeled ODN at the 4 h time point, compared with control, in the presence of 0.1 μM dendrimer. At the 24 h time point, these cells showed only a 2- to 3-fold increase in uptake and/or retention of 32P-labeled ODN in the presence of the dendrimer, compared with the control. In contrast, MCF-7 cells showed a maximal increase of 11-fold over the controls at the 6 h time point, and the efficacy was retained at 7-fold over the controls at the 24 h time point.
Figure 5. Cell-specific differences in the uptake of 32P-labeled ODN in the presence of G-4 dendrimer. Time-dependent ODN uptake in MCF-7 and SK-BR-3 breast cancer cells, the LNCaP prostate cancer cell line and the SK-OV-3 ovarian cancer cell line is shown. The experiments were performed as described in the legend to Figure 2.
Effects of dendrimers on cell viability
We further examined the effects of different dendrimers on the viability of MDA-MB-231 cells in order to determine the toxicity of the dendrimers. Cell viability was determined by trypan blue exclusion assay after 1–5 days of treatment. Table 2 shows the percentage of viable cells at different time points after dosing with dendrimers of different generations. Dendrimers of G-1 to G-4 had no significant effect on cell viability up to 0.5 μM concentrations even after 5 days of treatment. G-5 dendrimer had no effect on cell viability up to 0.25 μM concentration; however, it reduced the percentage of viable cells at 0.5 μM concentration. All the five generations of dendrimers were found to be toxic above 0.5 μM concentrations. G-5 dendrimer was found to be the most toxic.
Table 2. MDA-MB-231 cell viability in the presence of different concentrations of dendrimers
Analysis of 32P-labeled ODN by PAGE
A major problem associated with ODNs for therapeutic use is their degradation by serum-derived endonuclease. Condensation of DNA by polyamines has been reported to protect DNA from degradation by DNase I (26). In order to test whether complexing of the ODN with the dendrimers could protect the ODN from degradation within the cell, we next conducted PAGE of the 32P-labeled ODN extracted from MDA-MB-231 cells at different time periods after treatment (Fig. 6). Lane 1 (C) of Figure 6 shows a control sample of 32P-labeled ODN, while lanes 2, 4, 6, 8 and 10 show samples extracted from cells treated with 32P-labeled ODN alone for 4, 6, 8, 24 and 48 h, respectively. Lanes 3, 5, 7, 9 and 11 show samples extracted from cells treated with 32P-labeled ODN–G-4 dendrimer complex. An 5- to 10-fold increase in intensity was observed after treatment with dendrimer complex of 32P-labeled ODN for 4–6 h. Treatment of cells with 32P-labeled ODN–G-4 complex for 8, 24 and 48 h showed a 12- to 16-fold increase in intensity of the extracted 32P-labeled ODN compared with that in cells treated with 32P-labeled ODN alone. A significant amount of 32P-labeled ODN was also found at the well of the gel, indicating aggregation of the extracted 32P-labeled ODN.
Figure 6. Stability of 32P-labeled ODN in MDA-MB-231 cells. Cells were treated with 32P-labeled ODN alone or 32P-labeled ODN–G-4 dendrimer complex for the indicated times, and washed with DNase I. 32P-Labeled ODN was extracted from cells, and characterized by 20% PAGE. Lanes 2, 4, 6, 8 and 10 show 32P-labeled ODN extracted from cells treated with 32P-labeled ODN alone. Lanes 3, 5, 7, 9 and 11 show 32P-labeled ODN extracted from cells treated with 32P-labeled ODN–G-4 complex. Lane 1 (C) is 32P-labeled ODN in sterile water, used as a marker of the intact 32P-labeled ODN migration in the gel.
Gel electrophoretic analysis was conducted before and after washing cells with DNase I to remove ODN that might be bound to the cell surface. Figure 6 shows a representative result from cells washed with DNase I. A high level of intact ODN was observed in 32P-labeled ODN–G-4 dendrimer-treated cells even after DNase I washing. These results indicate that the increased level of 32P-labeled ODN extracted from treated cells is due to increased cellular uptake and retention in the presence of the dendrimer.
Effects of ODN, dendrimers and ODN–dendrimer complex on cell proliferation
The ODN used in the present series of experiments is a triplex-forming oligonucleotide targeted to the c-myc oncogene (22,27). Since c-myc is involved in cell proliferation, we next determined the effects of the ODN and G-4 dendrimer as single agents and in combination on the proliferation of MDA-MB-231 cells, using thymidine incorporation assay. Figure 7 shows the effects of different concentrations of ODN alone on DNA synthesis at 24, 48 and 72 h of treatment. At 0.1 and 0.25 μM ODN, there was no significant change in DNA synthesis at 24, 48 or 72 h of treatment. In contrast, at 0.5 μM ODN, DNA synthesis was reduced to 66 and 43% of the control, respectively, at 24 and 48 h of treatment. Increasing the concentration of ODN to 1 μM reduced DNA synthesis to 40% of the control at 24, 48 and 72 h of treatment.
Figure 7. Effect of different concentrations of ODN on MDA-MB-231 cells, as determined by thymidine incorporation assay. Thymidine incorporation assay was conducted after treating cells with ODN for 1, 2 or 3 days. The asterisks indicate significantly different from the untreated control group. P < 0.05; n = 6. Statistical significance was calculated using one-way ANOVA.
Since high concentrations (0.5 and 1 μM) of the ODN can form G-quartets that can produce cell growth-inhibitory effects, we next examined if low concentrations (0.1 and 0.25 μM) of the ODN can act in concert with G-4 to inhibit cell growth. Figure 8A shows the effect of ODN and G-4 on the proliferation of MDA-MB-231 cells. At 0.1 and 0.25 μM ODN or 0.1 and 0.25 μM dendrimer concentrations, there was no significant decrease in DNA synthesis. In contrast, there was up to a 40% decrease in DNA synthesis by a combination of 0.1 μM of ODN and 0.25 μM of G-4 dendrimer at 24 h of treatment. An 30–40% decrease in thymidine uptake was observed for all the ODN–G-4 dendrimer combinations tested at 24, 48 and 72 h of treatment, indicating that the biological activity of the ODN is facilitated by its complexation with the dendrimer.
Figure 8. (A) Effects of different concentrations of G-4 dendrimer and G-4 dendrimer–31 nt ODN complex on MDA-MB-231 cell growth, as determined by thymidine incorporation assay. The asterisks indicate that thymidine incorporation in these groups is significantly different from the control groups (P < 0.05; n = 6). Statistical significance was calculated using one-way ANOVA. (B) Effects of different concentrations of G-4 dendrimer and reversed orientation 31 nt ODN on MDA-MB-231 cell growth as single agents and in combination, as determined by thymidine incorporation assay. There was no significant difference (P > 0.05; n = 6) between control and treatment groups.
In order to test the possibility that the ODN–G-4-mediated reduction in thymidine incorporation may be due to G-quartet formation of the ODN, we performed control experiments using reversed orientation 31 nt ODN which is capable of forming quartet, but unable to form triplex at the c-myc promoter region. In these control experiments, an 20% decrease in thymidine incorporation was observed in cells treated with reversed orientation ODN–G-4 complex at the 24 h time point, compared with that of control (Fig. 8B). This decrease was not statistically significant (P > 0.05; n = 6). In addition, there was no significant difference in thymidine incorporation between the control and treatment groups at the 48 and 72 h time points. This result indicates a certain degree of specificity in the action of the triplex-forming ODN in cell growth inhibition.
Western blot analysis for c-myc protein
In the next set of experiments, we determined the effects of the triplex-forming ODN and G-4 dendrimer as single agents and in combination on c-myc protein levels in MDA-MB-231 cells. At 0.1–0.25 μM ODN or 0.1–0.25 μM dendrimer concentrations, there was no significant decrease in c-myc protein levels (Fig. 9). In contrast, there was a significant (P < 0.01, n = 3) decrease in c-myc protein levels in cells treated for 72 h with ODN–G-4 complex. A 65% decrease in c-myc protein level occurred in cells treated with 0.25 μM ODN and 0.25 μM G-4 dendrimer.
Figure 9. Effect of ODN and G-4 dendrimer as single agents and in combination on c-myc protein levels in MDA-MB-231 cells. Cells were treated for 72 h, protein extracted and c-myc level determined by western blot analysis. A representative result from western blot analysis is shown in the upper panel. Similar results were obtained in three separate experiments. The ? actin level was probed to confirm uniformity of protein loading. The lower panel shows the mean ± SD of the intensity of c-myc protein bands from three separate experiments. *P < 0.01 (n = 3) compared with control.
Particle size of ODN nanoparticles
It has been suggested that DNA uptake is related to nanoparticle formation in the presence of DNA delivery vehicles (1,28–31). Although considerable work has been done on the physical chemical aspects of DNA condensation in the presence of various agents, relatively little information is available on nanoparticle formation by ODNs. A recent study by Dauty et al. (32) showed the ability of ODNs to form DNA nanoparticles. Therefore, we next examined whether ODN nanoparticle formation occurred in the presence of the PPI dendrimers when the 31 nt ODN was complexed with these molecules. The dynamic laser light scattering technique was used to detect the presence of nanoparticles and measure their hydrodynamic radii. Since cellular uptake experiments were conducted in cell culture media, which contain physiological levels of salts, the particle size measurements were done in a buffer containing physiologically compatible salt concentrations (120 mM KCl, 10 mM NaCl, 2 mM MgCl2 and 0.1 mM CaCl2). In our experiments, nanoparticles were detected in ODN solution in the presence of the dendrimers. The dendrimers alone or ODN alone showed values of <0.1nm for the hydrodynamic radius, indicating that under the conditions of our experiments, these molecules did not undergo measurable self-aggregation. However, nanoparticles were observed when the ODN was complexed with the dendrimers, and the hydrodynamic radii of the particles were in the range 130–280. Table 3 shows the size of particles formed from ODN, after complexing it with different concentrations of dendrimers. G-4 and G-5 dendrimers produced the smallest particles. No ODN particle was detected with 1 and 2.5 μM concentrations of G-1 and G-2 dendrimers.
Table 3. Hydrodynamic radius of the nanoparticles formed from 31 nt ODN in the presence of PPI dendrimersa
DISCUSSION
Our data demonstrate the feasibility of using PPI dendrimers for improved delivery of a potential therapeutic ODN in several cancer cell lines in culture. Complexation of 32P-labeled ODN with PPI dendrimers of all five generations enhanced the internalization of the 32P-labeled ODN, compared with uncomplexed 32P-labeled ODN in MDA-MB-231 cells. Although optimal 32P-labeled ODN uptake differs with the cell line, the PPI dendrimers studied herein are capable of facilitating the uptake of the 32P-labeled ODN in all five cell lines tested (MDA-MB-231, MCF-7, SK-BR-3, SKOV-3 and LNCaP). The efficiency of dendrimers in enhancing 32P-labeled ODN uptake is dependent on the molecular weight of the dendrimer. This might be due to differences in the number of amino groups, flexibility of the dendrimer or the structural features of the condensed ODN. Among the dendrimers studied, G-4 was the most efficient in facilitating ODN internalization. A steady increase in ODN uptake was observed in MDA-MB-231 cells, with increase in molecular weight, up to the fourth generation dendrimer, and then there was a decrease in ODN uptake for G-5.
The concentration of the dendrimer was important for the cellular internalization of the ODN. Uptake increased with dendrimer concentration, reached a maximum at 0.05 μM concentration for G-4 dendrimer and then leveled off. The rapid increase in uptake with increase in dendrimer concentration may be due to charge reversal of the complex from negative to positive. Nguyen and Shklovskii (33) proposed charge reversal of DNA resulting from the fractionalization of positive charges, driven by the adsorption of additional polyelectrolyte molecules. On approaching the neutralized DNA, a new polyelectrolyte molecule can invade the DNA by detaching a portion of the attached polymer. The invasion of the new molecule on the surface of the neutral complex results in the formation of a complex with net positive charge. This net positive charge might help to generate a uniform distribution of particles by repulsion of positive charges. A recent atomic force microscopic study has shown DNA nanoparticle formation in the presence of PAMAM dendrimers with some degree of uniformity (34). In our study, cellular uptake of ODN–dendrimer complexes was facile over a wide range of N/P ratios (ratio of the number of terminal amino groups in the dendrimer to the number of phosphate groups in the ODN). This is exemplified by the uptake of 32P-labeled ODN (0.4 nM) at an N/P ratio of 133 and of unlabeled ODN (0.25 μM) at an N/P ratio of 1, used for growth inhibition and western blot studies. The efficacy of uptake at this concentration of ODN was confirmed by mixing 32P-labeled ODN with unlabeled ODN and conducting the uptake studies (results not presented). However, the requirement for a high N/P ratio at the low ODN concentration may also indicate ion competition in the cell culture medium and the possibility that dendrimers may not be fully charged under these conditions (35) and/or steric hindrance in ODN–dendrimer interactions. These different possibilities will be addressed in future studies.
Results of PAGE experiments provide direct evidence for the increased stability of ODN in the presence of the dendrimer. Complexation with dendrimer markedly increased the availability of intact 32P-labeled ODN within the cells compared with uncomplexed 32P-labeled ODN. We tested the possible presence of ODN–G-4 clusters at the cell surface by washing the cells with DNase I solution. We found that a high level of ODN was retained in the cellular extract even after this washing (Fig. 6). This finding is important since the therapeutic success of gene delivery depends on the availability and retention of intact DNA within the cell for prolonged periods of time. The concentration of ODN required for inhibition of DNA synthesis decreased when ODN was complexed with G-4 dendrimer. In contrast, an ODN with reversed orientation did not cause a significant growth inhibition at 24, 48 or 72 h of treatment (Fig. 8B). In addition, western blot analysis showed a significant 65% decrease in c-myc protein level in cells treated with the triplex-forming oligonucleotide and G-4 (Fig. 9). Although these results are not sufficient proof for a triplex DNA mechanism, collectively they support the hypothesis that the triplex-forming oligonucleotide can exert sequence specificity in cell growth inhibition and c-myc protein expression.
A recent study by Zinselmeyer et al. (21) shows that PPI dendrimers can be used for the delivery of a plasmid DNA. In contrast to our observation, their report showed maximal uptake with G-2 dendrimer. The greater conformational mobility of the long DNA molecule may facilitate its interaction with the extended structure of lower generation dendrimers more efficiently than the interaction of these dendrimers with the 31 nt ODN used in our study. They also found that the number of binding sites between DNA and the dendrimer increased with molecular weight. It is possible that for a long DNA molecule, sufficient binding sites may be achieved with lower generation dendrimers compared with small ODNs. Thus, the size of the ODN/DNA might be an important factor in optimizing the efficiency of the dendrimer.
Uptake enhancement of ODN by dendrimers is perceived to be a consequence of their ability to form tight complexes with the ODN (36). The nanoscopic structures may pass through the lipid cellular membrane more easily than large particles. Because of the high local functional group and compact structure, the dendrimer can incorporate a large number of ODN on the surface and also interior tertiary amines. Evans et al. (36) recently reported structural polymorphism of DNA–dendrimer complexes using synchrotron X-ray diffraction and polarizing microscopic studies. An analogous situation is our recent findings of multiple liquid crystalline textures in polyamine-complexed calf thymus DNA (37). In our studies, the PPI dendrimer was found to condense the ODN, a phenomenon more commonly investigated with low molecular weight cations, such as spermine (1,25).
Zinselmeyer et al. (21) showed that the biocompatibility and transfection activity of G-2 and G-3 dendrimers were equivalent to commercial DOTAP. The size of the DNA–dendrimer complex determined by photon correlation spectroscopy in their experiment was between 163 and 518 nm. Data obtained for the ODN–dendrimer complex by dynamic light scattering experiments in our study showed a size of 130–280 nm, indicating that the size of the complex is not much affected by the chain length of ODN/DNA. No aggregation was observed at the physiologically compatible buffer conditions with 130 mM Na+/K+ ions, as particle size measurement was conducted in this buffer. The size of ODN nanoparticles was similar in a buffer containing 150 mM NaCl (unpublished results).
Haensler and Sz?ka (19) proposed that dendrimers with surface and internal amino groups can act as endosomal buffering agents similar to PEI, and can cause endosomal rupture, releasing the complex to the cytoplasm. Tang and Sz?ka (38) hypothesized that uncomplexed dendrimers adopt an extended conformation because of the repulsion between the charged surface amino groups. On complexation, the dendrimer collapses to a compact form because of charge neutralization (37). In the endosomal compartment, the interior tertiary amine becomes protonated, and hence less polymer is required to maintain charge neutralization. Excess polymer is then released and swells, causing endosomal rupture and release of the complex into the cytoplasm. The therapeutic efficacy of ODN depends on the intracellular fate and availability of ODN for hybridization with the target site. This can occur only when the ODN is free or loosely bound to the vector. Yoo and Juliano (39) found that unlike cationic lipid delivery agents, where the separation of the nucleic acid and delivery agent occurs in endosomes, the dendrimer–ODN complex remains intact as it enters the nucleus. Belinska et al. (40) reported that binding to dendrimer does not interfere with the ability of DNA to form hydrogen bonds with the complementary sequence in the target RNA or gene and that ODN detachment from the dendrimer is not essential for its action.
A major concern with the use of dendrimers as vectors for ODN delivery is their toxicity. The cytotoxicity of cationic dendrimers is believed to be due to the interaction between the positively charged dendrimer and negatively charged cellular structures (41,42). Malik et al. (42) reported the in vitro biocompatibility of G-2, G-3 and G-4 PPI dendrimers. The cytotoxicity measured by them showed an IC50 value of 0.3 mg/ml for B16F10 cells by MTT assay for G-2, 0.05 mg/ml for G-3, and 0.05 mg/ml for G-4 dendrimers. Lim et al. (43) reported that the cytotoxicity of PPI dendrimer is lower than that of PEI in Vero cells by MTT assay. The cell viability in our studies showed no toxicity for dendrimers of generations 1 to 5 under the concentrations used in ODN uptake and cell proliferation studies. Zinselmeyer et al. (21) found molecular weight-dependent toxicity by the MTT assay against a human epidermoid carcinoma cell line. In addition, Shah et al (44) found that the toxicity of dendrimers was reduced on complexation with DNA. They constructed an amphipathic asymmetric dendrimer and studied its transfection to BHK-21 cells in a complex with plasmid DNA. BHK cells were found to exhibit greater survival in the presence of the dendrimer–DNA complex than when dendrimer was applied alone. ODN–dendrimer complex has been shown to be less toxic than unmodified ODN, possibly due to the reduced charge of the complex (39,45). PPI dendrimer–gadolinium (Gd) complex has been reported to be useful as a magnetic resonance contrast agent (46,47). Toxicological studies of PPI G-5 dendrimer–Gd complex in mouse showed no significant adverse effect on body weight and organs compared with control. The mouse was injected with 0.15 mM of Gd/kg of the complex within 10 weeks (47).
Conclusions
In summary, this study shows that PPI dendrimers are highly efficient in enhancing the uptake of ODN by cells at non-toxic concentrations. Differences in efficiency with increasing generations of dendrimers suggest that the increased uptake is dependent on the charge density and flexibility of the dendrimer. Charge density increases and flexibility decreases with the increasing generation of the dendrimer. Optimal efficiency is obtained with G-4 dendrimer for the 31 nt ODN. The enhanced availability of intact ODN within cells up to 48 h or more and the improved antiproliferative effect of ODN on complexation with this dendrimer have important implications in ODN-based therapeutics. The high surface charge density of the polymer facilitates chemical modification. Assembling biologically interesting molecules such as amino acids, folate receptors, sugar derivatives, etc. at the dendrimer periphery presents an attractive avenue of research to improve the properties of this carrier. The versatility of its properties, ease of chemical modification and high cellular uptake of the complex by different cancer cell lines make PPI dendrimers an important new entry as an ODN/DNA delivery vehicle for gene therapy.
ACKNOWLEDGEMENTS
This work was supported, in part, by Public Health Service grants CA80163, CA73058 and CA42439 from the National Cancer Institute, and by a Susan G. Komen Breast Cancer Research Foundation Grant Award.
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*To whom correspondence should be addressed at UMDNJ-Robert Wood Johnson Medical School, 125 Paterson Street, CAB 7090, New Brunswick, NJ 08903, USA. Tel: +1 732 235 8460; Fax: +1 732 235 8473; Email: thomastj@umdnj.edu
ABSTRACT
We used polypropylenimine dendrimers for delivering a 31 nt triplex-forming oligonucleotide (ODN) in breast, prostate and ovarian cancer cell lines, using 32P-labeled ODN. Dendrimers enhanced the uptake of ODN by 14-fold in MDA-MB-231 breast cancer cells, compared with control ODN uptake. Dendrimers exerted their effect in a concentration- and molecular weight-dependent manner, with generation 4 (G-4) dendrimer having maximum efficacy. A similar increase in ODN uptake was found with MCF-7 and SK-BR-3 (breast), LNCaP (prostate) and SK-OV-3 (ovarian) cancer cells. The dendrimers had no significant effect on cell viability at concentrations at which maximum ODN uptake occurred. Thymidine incorporation showed that complexing the ODN with G-4 significantly increased the growth-inhibitory effect of the ODN. Western blot analysis showed a significant 65% reduction of c-myc protein level in ODN–G-4 treated cells compared with that of ODN-treated/control cells. Gel electrophoretic analysis showed that ODN remained intact in cells even after 48 h of treatment. The hydrodynamic radii of nanoparticles formed from ODN in the presence of the dendrimers were in the range of 130–280 nm, as determined by dynamic laser light scattering. Taken together, our results indicate that polypropylenimine dendrimers might be useful vehicles for delivering therapeutic oligonucleotides in cancer cells.
INTRODUCTION
Gene therapy relies on the efficient transport of oligonucleotides and plasmid DNA through the cell membrane by mechanisms that are not well defined at present (1). Therapeutic oligonucleotides (ODNs) offer the possibility of disrupting the expression of disease-related genes using short synthetic nucleic acid sequences (2–4). Gene expression can be disrupted at the transcriptional (triplex DNA) or translational (antisense DNA) level. In the triplex DNA-based anti-gene approach, transcription is disrupted by the binding of a triplex-forming oligonucleotide at the promoter region of a target gene (5). In contrast, antisense ODN is designed to specifically bind to short complementary sequence of target mRNA in order to prevent the translation of the target gene (6). The ODN is expected to hybridize with the mRNA and block either translational initiation or elongation of transcripts. Because of the high affinity and selectivity of these ODNs for their target sites, they are expected to exert a high level of specificity, and hence represent an interesting class of drugs. Despite this theoretical scenario, there are several limitations in advancing ODN therapeutics to the clinic. For the clinical applications, an important criterion is the availability of a suitable carrier that can efficiently transport and release the ODN at the desired intracellular sites at a controlled rate (7).
There has been much interest in developing new formulations and materials for delivering ODNs to cellular targets in vitro and in vivo. Viral delivery systems have been used in many applications and clinical trials; however, the immune response to viral proteins continues to be a daunting problem (8,9). Lipid-based systems represent a promising class of gene carriers; however, their toxicity on repeated dosing and poor in vivo performance are major drawbacks in advancing them to the clinic (10). There has been considerable interest in recent years in developing polymeric material, such as polyethylenimine (PEI), as gene delivery vehicles (11). PEI is efficient in gene delivery both in vitro and in vivo (12,13). An advantage of PEI is that it allows efficient gene transfer without the need for agents facilitating endosomal escape. Protonated amino groups of PEI appear to buffer acidic endosomal compartment and release DNA to the cytoplasm (14). The transfection efficiency and cytotoxicity of this polymer are dependent on its molecular weight and polydispersity (15,16).
While polymers such as PEI have a range of molecular weight distribution, the novel highly branched three-dimensional molecules, called dendrimers, have defined molecular weight and a large number of controllable peripheral functionalities (17). Tomalia et al. (18) reported the first preparation of an entire series of dendrimers possessing trigonal, 1- > 2 N-based, branching centers. Compared with linear and branched polymers, the monodispersity and controllable surface functionality make the dendrimers an interesting class of gene delivery vehicles. Of the vast majority of dendrimers synthesized, only a few are water soluble and non-toxic, thus rendering them useful to the pharmaceutical industry. Haensler and Sz?ka (19) first showed that polyamidoamine (PAMAM) dendrimers were efficient gene transfer agents for a variety of cultured mammalian cells. Polypropylenimine (PPI) dendrimers, DAB-dendr-(NH2)x, contain terminal amino groups similar to PAMAM. Repetitive addition of a primary amine to two equivalents of acrylonitrile, followed by catalytic hydrogenation, produces polyamine-terminated PPI dendrimers (17,20). A recent report showed that generation 2 and generation 3 PPI dendrimers were effective in transfecting a plasmid DNA in A431 cells (21). In the present study, we investigated the efficacy of five generations of PPI dendrimers to deliver a 31 nt triplex-forming oligonucleotide targeted to the c-myc oncogene (22) in breast, prostate and ovarian cancer cell lines. Our results show that the dendrimers are capable of facilitating the uptake of the ODN in several cancer cell lines in a concentration- and structure-dependent manner.
MATERIALS AND METHODS
Oligonucleotide
HPLC-purified 31 nt phosphodiester ODN was purchased from Oligos, Etc. (Wilsonville, OR). The sequence of the triplex-forming ODN used in our study is: d(GTGGTGG GGTGGTTGGGGTGGGTGGGGTGGG). This sequence is targeted to a triplex-forming region in the promoter of the c-myc oncogene (22,23). As a control, we used d(GGGT GGGGTGGGTGGGGTTGGTGGGGTGGTG), a 31 nt ODN with reversed orientation that could still form G-quartets. The triplex-forming 31 nt ODN was 5'-end labeled with ATP using T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN). Unincorporated label was removed by passing the solution through a Chroma Spin Column (Clontech Lab, Inc., Palo Alto, CA).
Dendrimers, chemicals and other reagents
Dendrimers, DAB Am-4 (polypropylenimine tetraamine dendrimer, generation-1; G-1), DAB Am-8 (polypropylenimine octaamine dendrimer, generation-2; G-2), DAB Am-16 (polypropylenimine hexadecaamine dendrimer, generation-3; G-3), DAB Am-32 (polypropylenimine dotriacontaamine dendrimer, generation-4; G-4) and DAB-Am-64 (polypropylenimine tetrahexacontaamine dendrimer generation-5; G-5) were purchased from Aldrich Chemical Co. (Milwaukee, WI). The chemical structures of the dendrimers are given in Figure 1. Fetal bovine serum (FBS), cell culture media, antibiotics and other chemicals were purchased from Sigma Chemical Co. (St Louis, MO). Monoclonal anti-c-myc antibody and secondary anti-IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used for western blot analysis of c-myc protein levels. Anti-?-actin monoclonal antibody was purchased from Sigma Chemical Co. DNase I was from Roche Diagnostics (Nutley, NJ).
Figure 1. Chemical structures of polypropylenimine dendrimers used in this study. The designations are: G-1, generation 1 dendrimer; G-2, generation 2 dendrimer; G-3, generation 3 dendrimer; G-4, generation 4 dendrimer; G-5, generation 5 dendrimer.
Cell culture
Three breast cancer cell lines (MDA-MB-231, MCF-7 and SK-BR-3), a prostate cancer cell line (LNCaP) and an ovarian cancer cell line (SKOV-3) were obtained from ATCC (Manassas, VA). MDA-MB-231 cells were maintained in minimal essential medium (MEM), supplemented with 10% FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamycin, 0.4 mM sodium pyruvate and 2 mM L-glutamine. MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamycin, 0.4 mM sodium pyruvate, 10 mM non-essential amino acids and 2 mM L-glutamine. Phenol red-free DMEM, containing 10% FBS , was used for experiments using MCF-7 cells to avoid the effects of serum-derived estrogenic compounds and phenol red (24). LNCaP cells were maintained in RPMI-1640 medium containing 10% serum, 10 mM HEPES, 1 mM sodium pyruvate and 2 mM L-glutamine. SK-BR-3 and SKOV-3 cells were grown in McCoy’s 5A medium containing 10% FBS and 3 mM L-glutamine.
Measurement of 32P-labeled ODN uptake
32P-Labeled ODN was added to different concentrations of dendrimer, mixed by vortexing for 5 min, and incubated at 22°C for 1 h. The final concentration of dendrimers ranged from 0.01 to 2.5 μM and the probe was used at 2.5 x 105 c.p.m. (0.4 nM). The complex was added to cells seeded in 6-well plates. Uptake of 32P-labeled ODN was determined as described below. Cells (5 x 105/well) were seeded in 2 ml of medium in 6-well plates and allowed to attach to the plates for 24 h (23). Prior to the experiment, the medium was removed and replaced with 0.5 ml per well of fresh medium, pre-warmed to 37°C. 32P-Labeled ODN (2.5 x 105 c.p.m. level) was added to the medium and the cells incubated at 37°C in a 5% CO2 incubator. The cells were harvested after 4, 6, 8 and 24 h of treatment. The medium was removed, and cells were washed three times with ice-cold phosphate-buffered saline (PBS). A 1 ml aliquot of 1 M NaOH was added to each sample and kept at 60°C for 30 min. The lysate was neutralized with 1 ml of 1 M HCl, and radioactivity quantified using a Beckman (LS 5000 TD) Scintillation Counter. ODN uptake was studied in MDA-MB-231, MCF-7, SK-BR-3, LNCaP and SKOV-3 cell lines.
Cell viability in the presence of dendrimer
Cell viability was determined after adding different concentrations of dendrimers to MDA-MB-231 cells. Cells were plated in 24-well plates at a density of 3.0 x 104 cells/well. Cells were dosed with different concentrations of the dendrimers of different generations. Re-dosing was done every 48 h, after replacing the medium with fresh medium. The number of viable cells was determined every 24 h for 5 days, using trypan blue exclusion assay. Cell viability was calculated as follows:
Cell viability = (no. of viable cells in the sample/no. of viable cells in the control) x 100.
PAGE of 32P-labeled ODN
MDA-MB-231 cells were incubated with 32P-labeled ODN or 32P-labeled ODN–G-4 dendrimer complex at 37°C for 48 h. Cells were harvested at 4, 6, 8, 24 and 48 h after treatment. Cells (in plates) were first washed three times with PBS and then incubated for 2 min with 300 U/ml of DNase I in 50 mM Tris–HCl pH 7.6 containing 10 mM MgCl2. Cells were then washed with PBS, trypsinized, collected in 1 ml of PBS, centrifuged at 1000 g for 5 min, and the supernatant removed. The cell pellet was lysed with 500 μl of 1% SDS lysis buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl and 1% SDS). The lysate was centrifuged at 3000 g for 15 min and supernatant collected in microcentrifuge tubes. ODN was precipitated by adding 50 μl of 5 M NaCl and 1 ml of cold 100% ethanol. After 1 h at –70°C, the solution was centrifuged at 12 000 g for 15 min. The supernatant was removed and the precipitate dried in air. Samples were dissolved in 50 μl of a solution containing 10 mM Tris–HCl and 50 mM NaCl, and analyzed by gel electrophoresis using a 20% polyacrylamide gel at 250 V for 3 h. The gel was dried, and exposed to X-ray film.
Thymidine incorporation assay
MDA-MB-231 cells were seeded in a 6-well plate at a density of 5 x 105 cells/well. After 24 h incubation at 37°C, triplicate wells were treated with ODN alone or ODN–dendrimer complex. ODN was complexed with dendrimer at 100-fold higher concentrations in order to obtain the desired final concentrations in the cell culture medium. DNA synthesis was quantified at 24, 48 and 72 h. For this purpose, 4 μCi/well of thymidine was added 1 h prior to the specified time points. After 1 h incubation at 37°C, cells were washed twice with ice-cold PBS and equilibrated with ice-cold 5% trichloroacetic acid (TCA) for 5 min. It was again washed twice with ice-cold 5% TCA. The cells were then dissolved in 1 M NaOH and neutralized with 1 M HCl. The radioactive thymidine incorporated in the cellular DNA was estimated by liquid scintillation counting.
As a control, the thymidine incorporation assay was repeated with the reversed orientation 31 nt ODN as a single agent or after complex formation with G-4 dendrimer.
Western blot analysis of c-myc protein
The levels of c-myc protein in control and ODN–G-4-treated MDA-MB-231 cells were determined by western blot analysis. MDA-MB-231 cells were seeded in 100 mm dishes at a density of 2 x 106 cells/well. After 24 h incubation at 37°C, cells were treated with ODN, G-4 dendrimer or ODN + G-4 dendrimer in combination. Cells were harvested after 72 h of treatment. Harvested cells were washed with PBS and homogenized by sonicating in lysis buffer containing 150 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 50 mM sodium fluoride, 0.2% SDS, 1 mM sodium vanadate, 2 μg/ml leupeptin, aprotonin and pepstatin, and 1 mM phenylmethylsulfonyl fluoride. A 30 μg aliquot of protein (determined by the Bradford protein assay) was diluted in 2x SDS–PAGE Laemmli buffer (150 mM Tris base pH 6.8, 30% glycerol, 4% SDS, 7.5 mM dithiothreitol, 0.01% bromophenol blue) and separated on a 10% SDS–polyacrylamide gel. After electrophoresis, separated proteins were transferred to PVDF immobilon membrane (Bedford, MA). The membrane was incubated in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, and a 1:200 dilution of monoclonal anti-c-myc antibody overnight. After washing, the membrane was incubated with a 1:2000 dilution of the secondary antibody for 1 h. Blots were then developed by the enhanced chemiluminescence detection technique. To verify equal protein loading, membranes were stripped and re-blotted with anti-? actin monoclonal antibody (1:5000). The intensity of the protein bands was quantified using Kodak 1D Image Analysis Software with a TWAIN Scanner. Lightly exposed films were used for quantification.
Particle size of ODN nanoparticles
The particle size of nanoparticles formed by complexing of ODN with PPI dendrimers was determined using dynamic laser light scattering equipment (25). Experiments were conducted using a DynaPro model MSX (Protein Solutions, Inc., Charlottesville, VA), equipped with a 2 W laser of 800 nm wavelength. Complexation of ODN with dendrimer was done in a buffer containing a physiological concentration of cations (120 mM KCl, 10 mM NaCl, 2 mM MgCl2 and 0.1 mM CaCl2). Then 40 μl of the solution was transferred to the standard quartz cuvette, and the hydrodynamic radii of the particles were determined using dynamic laser light scattering at an angle of 90° at 22°C (25). Data analysis was performed using a Dynamics Version 6 software package supplied by the manufacturer.
RESULTS
Uptake of ODN by MDA-MB-231 cells
Dendrimers of five generations, which differed in molecular weight and number of peripheral amino groups, were used for the uptake study. 32P-Labeled ODN (2.5 x 105 c.p.m./well) was mixed with different concentrations of dendrimers in sterile water. The 32P-labeled ODN–dendrimer complex was added to MDA-MB-231 cells in culture and incubated for 4, 6, 8 or 24 h. The amount of 32P-labeled ODN taken up by cells was quantified by scintillation counting (23). Figure 2 shows the uptake of 32P-labeled ODN after complexing it with G-4 dendrimer. There was a concentration- and/or time-dependent increase in uptake for both control and dendrimer-treated samples. The increase in 32P-labeled ODN uptake was 14-fold higher than that of control, in the presence of 0.1–0.25 μM concentrations of dendrimer at 4 h of treatment, and the enhanced uptake was maintained even after 24 h of treatment. However, there was a decrease in 32P-labeled ODN uptake at the 24 h time point.
Figure 2. Cellular uptake of 32P-labeled ODN by MDA-MB-231 cells, after complexing the ODN with G-4 dendrimer. Cells were treated with 250 000 c.p.m. (0.4 nM) of 32P-labeled ODN after complexing it with G-4 dendrimer in 6-well plates. At the indicated times, the medium was removed, cells washed three times with cold PBS, lysed and cell-associated radioactivity quantified by scintillation counting. Control indicates the use of labeled ODN in the absence of dendrimer. The percentage increase in uptake is calculated with reference to the control group. Results presented are the mean of three separate triplicate measurements. Error bars indicate the SEM.
Figure 3 shows the effects of different generations of dendrimers on 32P-labeled ODN uptake after cells were treated with 32P-labeled ODN complexed with dendrimers. In all cases, there was a concentration-dependent increase in 32P-labeled ODN uptake up to 0.1 μM dendrimer concentration, and then the effect leveled off. G-4 dendrimer was the most efficacious, showing a 14-fold increase in 32P-labeled ODN uptake compared with control. G-5 dendrimer was less efficient than G-4, giving a maximum 8-fold increase compared with control. Table 1 presents the concentrations required for maximal 32P-labeled ODN uptake, using different generations of dendrimers. G-4 dendrimer exerted optimum efficiency at 0.05 μM concentration.
Figure 3. Concentration-dependent increase in the cellular uptake of 32P-labeled ODN in MDA-MB-231 cells in the presence of different dendrimers. Cells were treated for 6 h with labeled ODN–dendrimer complex. The symbols are as follows: G-1 dendrimer, closed circle; G-2 dendrimer, inverted open triangle; G-3 dendrimer, closed square; G-4 dendrimer; open diamond; and G-5 dendrimer, closed triangle. Results presented are the mean of three separate triplicate measurements. Error bars indicate the SEM. In some cases, the error bars are smaller than the size of the symbols.
Table 1. Concentrations of polypropylenimine dendrimers that caused maximum uptake of 32P-labeled ODN in MDA-MB-231 cells
Figure 4 shows the time-dependent increase in 32P-labeled ODN uptake at 0.1 μM concentration of the dendrimers. The efficiency of the dendrimers in facilitating 32P-labeled ODN uptake increased with molecular weight of the dendrimer, except for G-5 dendrimer. Uptake efficiency remained high for G-4 dendrimer at 4, 6 and 8 h, but decreased at the 24 h time point.
Figure 4. Uptake of 32P-labeled ODN in MDA-MB-231 cells after complexing it with a fixed concentration (0.1 μM) of G-1, G-2, G-3, G-4 or G-5 dendrimers. Cells were treated with the indicated dendrimer–ODN complex, and harvested at 4, 6, 8 or 24 h. Results presented are the mean ± SEM.
Uptake of 32P-labeled ODN by other cancer cell lines
In order to examine whether the effectiveness of dendrimers in increasing the uptake of the 31 nt ODN is cell type specific, we next studied the ability of the dendrimers to alter the uptake of the 32P-labeled ODN in SK-BR-3, MCF-7, LNCaP and SKOV-3 cells. Figure 5 shows the uptake of ODN by different cell lines after complexing the ODN with G-4 dendrimer. Although there were cell-specific differences in the maximal uptake of the ODN, the dendrimers enhanced the uptake of the 31 nt ODN in all cell lines tested. There was a 23-fold increase in 32P-labeled ODN uptake compared with control in the ovarian cancer cell line, SK-OV-3, at the 4 h time point in the presence of 0.5 μM dendrimer. However, the effectiveness of the dendrimer on the uptake and/or retention of 32P-labeled ODN decreased with time, demonstrating only a 6-fold increase at the 24 h time point, compared with the control. In SK-BR-3 breast cancer cells, there was a 19-fold increase in the uptake of the 32P-labeled ODN at the 4 h time point, compared with control, in the presence of 0.1 μM dendrimer. At the 24 h time point, these cells showed only a 2- to 3-fold increase in uptake and/or retention of 32P-labeled ODN in the presence of the dendrimer, compared with the control. In contrast, MCF-7 cells showed a maximal increase of 11-fold over the controls at the 6 h time point, and the efficacy was retained at 7-fold over the controls at the 24 h time point.
Figure 5. Cell-specific differences in the uptake of 32P-labeled ODN in the presence of G-4 dendrimer. Time-dependent ODN uptake in MCF-7 and SK-BR-3 breast cancer cells, the LNCaP prostate cancer cell line and the SK-OV-3 ovarian cancer cell line is shown. The experiments were performed as described in the legend to Figure 2.
Effects of dendrimers on cell viability
We further examined the effects of different dendrimers on the viability of MDA-MB-231 cells in order to determine the toxicity of the dendrimers. Cell viability was determined by trypan blue exclusion assay after 1–5 days of treatment. Table 2 shows the percentage of viable cells at different time points after dosing with dendrimers of different generations. Dendrimers of G-1 to G-4 had no significant effect on cell viability up to 0.5 μM concentrations even after 5 days of treatment. G-5 dendrimer had no effect on cell viability up to 0.25 μM concentration; however, it reduced the percentage of viable cells at 0.5 μM concentration. All the five generations of dendrimers were found to be toxic above 0.5 μM concentrations. G-5 dendrimer was found to be the most toxic.
Table 2. MDA-MB-231 cell viability in the presence of different concentrations of dendrimers
Analysis of 32P-labeled ODN by PAGE
A major problem associated with ODNs for therapeutic use is their degradation by serum-derived endonuclease. Condensation of DNA by polyamines has been reported to protect DNA from degradation by DNase I (26). In order to test whether complexing of the ODN with the dendrimers could protect the ODN from degradation within the cell, we next conducted PAGE of the 32P-labeled ODN extracted from MDA-MB-231 cells at different time periods after treatment (Fig. 6). Lane 1 (C) of Figure 6 shows a control sample of 32P-labeled ODN, while lanes 2, 4, 6, 8 and 10 show samples extracted from cells treated with 32P-labeled ODN alone for 4, 6, 8, 24 and 48 h, respectively. Lanes 3, 5, 7, 9 and 11 show samples extracted from cells treated with 32P-labeled ODN–G-4 dendrimer complex. An 5- to 10-fold increase in intensity was observed after treatment with dendrimer complex of 32P-labeled ODN for 4–6 h. Treatment of cells with 32P-labeled ODN–G-4 complex for 8, 24 and 48 h showed a 12- to 16-fold increase in intensity of the extracted 32P-labeled ODN compared with that in cells treated with 32P-labeled ODN alone. A significant amount of 32P-labeled ODN was also found at the well of the gel, indicating aggregation of the extracted 32P-labeled ODN.
Figure 6. Stability of 32P-labeled ODN in MDA-MB-231 cells. Cells were treated with 32P-labeled ODN alone or 32P-labeled ODN–G-4 dendrimer complex for the indicated times, and washed with DNase I. 32P-Labeled ODN was extracted from cells, and characterized by 20% PAGE. Lanes 2, 4, 6, 8 and 10 show 32P-labeled ODN extracted from cells treated with 32P-labeled ODN alone. Lanes 3, 5, 7, 9 and 11 show 32P-labeled ODN extracted from cells treated with 32P-labeled ODN–G-4 complex. Lane 1 (C) is 32P-labeled ODN in sterile water, used as a marker of the intact 32P-labeled ODN migration in the gel.
Gel electrophoretic analysis was conducted before and after washing cells with DNase I to remove ODN that might be bound to the cell surface. Figure 6 shows a representative result from cells washed with DNase I. A high level of intact ODN was observed in 32P-labeled ODN–G-4 dendrimer-treated cells even after DNase I washing. These results indicate that the increased level of 32P-labeled ODN extracted from treated cells is due to increased cellular uptake and retention in the presence of the dendrimer.
Effects of ODN, dendrimers and ODN–dendrimer complex on cell proliferation
The ODN used in the present series of experiments is a triplex-forming oligonucleotide targeted to the c-myc oncogene (22,27). Since c-myc is involved in cell proliferation, we next determined the effects of the ODN and G-4 dendrimer as single agents and in combination on the proliferation of MDA-MB-231 cells, using thymidine incorporation assay. Figure 7 shows the effects of different concentrations of ODN alone on DNA synthesis at 24, 48 and 72 h of treatment. At 0.1 and 0.25 μM ODN, there was no significant change in DNA synthesis at 24, 48 or 72 h of treatment. In contrast, at 0.5 μM ODN, DNA synthesis was reduced to 66 and 43% of the control, respectively, at 24 and 48 h of treatment. Increasing the concentration of ODN to 1 μM reduced DNA synthesis to 40% of the control at 24, 48 and 72 h of treatment.
Figure 7. Effect of different concentrations of ODN on MDA-MB-231 cells, as determined by thymidine incorporation assay. Thymidine incorporation assay was conducted after treating cells with ODN for 1, 2 or 3 days. The asterisks indicate significantly different from the untreated control group. P < 0.05; n = 6. Statistical significance was calculated using one-way ANOVA.
Since high concentrations (0.5 and 1 μM) of the ODN can form G-quartets that can produce cell growth-inhibitory effects, we next examined if low concentrations (0.1 and 0.25 μM) of the ODN can act in concert with G-4 to inhibit cell growth. Figure 8A shows the effect of ODN and G-4 on the proliferation of MDA-MB-231 cells. At 0.1 and 0.25 μM ODN or 0.1 and 0.25 μM dendrimer concentrations, there was no significant decrease in DNA synthesis. In contrast, there was up to a 40% decrease in DNA synthesis by a combination of 0.1 μM of ODN and 0.25 μM of G-4 dendrimer at 24 h of treatment. An 30–40% decrease in thymidine uptake was observed for all the ODN–G-4 dendrimer combinations tested at 24, 48 and 72 h of treatment, indicating that the biological activity of the ODN is facilitated by its complexation with the dendrimer.
Figure 8. (A) Effects of different concentrations of G-4 dendrimer and G-4 dendrimer–31 nt ODN complex on MDA-MB-231 cell growth, as determined by thymidine incorporation assay. The asterisks indicate that thymidine incorporation in these groups is significantly different from the control groups (P < 0.05; n = 6). Statistical significance was calculated using one-way ANOVA. (B) Effects of different concentrations of G-4 dendrimer and reversed orientation 31 nt ODN on MDA-MB-231 cell growth as single agents and in combination, as determined by thymidine incorporation assay. There was no significant difference (P > 0.05; n = 6) between control and treatment groups.
In order to test the possibility that the ODN–G-4-mediated reduction in thymidine incorporation may be due to G-quartet formation of the ODN, we performed control experiments using reversed orientation 31 nt ODN which is capable of forming quartet, but unable to form triplex at the c-myc promoter region. In these control experiments, an 20% decrease in thymidine incorporation was observed in cells treated with reversed orientation ODN–G-4 complex at the 24 h time point, compared with that of control (Fig. 8B). This decrease was not statistically significant (P > 0.05; n = 6). In addition, there was no significant difference in thymidine incorporation between the control and treatment groups at the 48 and 72 h time points. This result indicates a certain degree of specificity in the action of the triplex-forming ODN in cell growth inhibition.
Western blot analysis for c-myc protein
In the next set of experiments, we determined the effects of the triplex-forming ODN and G-4 dendrimer as single agents and in combination on c-myc protein levels in MDA-MB-231 cells. At 0.1–0.25 μM ODN or 0.1–0.25 μM dendrimer concentrations, there was no significant decrease in c-myc protein levels (Fig. 9). In contrast, there was a significant (P < 0.01, n = 3) decrease in c-myc protein levels in cells treated for 72 h with ODN–G-4 complex. A 65% decrease in c-myc protein level occurred in cells treated with 0.25 μM ODN and 0.25 μM G-4 dendrimer.
Figure 9. Effect of ODN and G-4 dendrimer as single agents and in combination on c-myc protein levels in MDA-MB-231 cells. Cells were treated for 72 h, protein extracted and c-myc level determined by western blot analysis. A representative result from western blot analysis is shown in the upper panel. Similar results were obtained in three separate experiments. The ? actin level was probed to confirm uniformity of protein loading. The lower panel shows the mean ± SD of the intensity of c-myc protein bands from three separate experiments. *P < 0.01 (n = 3) compared with control.
Particle size of ODN nanoparticles
It has been suggested that DNA uptake is related to nanoparticle formation in the presence of DNA delivery vehicles (1,28–31). Although considerable work has been done on the physical chemical aspects of DNA condensation in the presence of various agents, relatively little information is available on nanoparticle formation by ODNs. A recent study by Dauty et al. (32) showed the ability of ODNs to form DNA nanoparticles. Therefore, we next examined whether ODN nanoparticle formation occurred in the presence of the PPI dendrimers when the 31 nt ODN was complexed with these molecules. The dynamic laser light scattering technique was used to detect the presence of nanoparticles and measure their hydrodynamic radii. Since cellular uptake experiments were conducted in cell culture media, which contain physiological levels of salts, the particle size measurements were done in a buffer containing physiologically compatible salt concentrations (120 mM KCl, 10 mM NaCl, 2 mM MgCl2 and 0.1 mM CaCl2). In our experiments, nanoparticles were detected in ODN solution in the presence of the dendrimers. The dendrimers alone or ODN alone showed values of <0.1nm for the hydrodynamic radius, indicating that under the conditions of our experiments, these molecules did not undergo measurable self-aggregation. However, nanoparticles were observed when the ODN was complexed with the dendrimers, and the hydrodynamic radii of the particles were in the range 130–280. Table 3 shows the size of particles formed from ODN, after complexing it with different concentrations of dendrimers. G-4 and G-5 dendrimers produced the smallest particles. No ODN particle was detected with 1 and 2.5 μM concentrations of G-1 and G-2 dendrimers.
Table 3. Hydrodynamic radius of the nanoparticles formed from 31 nt ODN in the presence of PPI dendrimersa
DISCUSSION
Our data demonstrate the feasibility of using PPI dendrimers for improved delivery of a potential therapeutic ODN in several cancer cell lines in culture. Complexation of 32P-labeled ODN with PPI dendrimers of all five generations enhanced the internalization of the 32P-labeled ODN, compared with uncomplexed 32P-labeled ODN in MDA-MB-231 cells. Although optimal 32P-labeled ODN uptake differs with the cell line, the PPI dendrimers studied herein are capable of facilitating the uptake of the 32P-labeled ODN in all five cell lines tested (MDA-MB-231, MCF-7, SK-BR-3, SKOV-3 and LNCaP). The efficiency of dendrimers in enhancing 32P-labeled ODN uptake is dependent on the molecular weight of the dendrimer. This might be due to differences in the number of amino groups, flexibility of the dendrimer or the structural features of the condensed ODN. Among the dendrimers studied, G-4 was the most efficient in facilitating ODN internalization. A steady increase in ODN uptake was observed in MDA-MB-231 cells, with increase in molecular weight, up to the fourth generation dendrimer, and then there was a decrease in ODN uptake for G-5.
The concentration of the dendrimer was important for the cellular internalization of the ODN. Uptake increased with dendrimer concentration, reached a maximum at 0.05 μM concentration for G-4 dendrimer and then leveled off. The rapid increase in uptake with increase in dendrimer concentration may be due to charge reversal of the complex from negative to positive. Nguyen and Shklovskii (33) proposed charge reversal of DNA resulting from the fractionalization of positive charges, driven by the adsorption of additional polyelectrolyte molecules. On approaching the neutralized DNA, a new polyelectrolyte molecule can invade the DNA by detaching a portion of the attached polymer. The invasion of the new molecule on the surface of the neutral complex results in the formation of a complex with net positive charge. This net positive charge might help to generate a uniform distribution of particles by repulsion of positive charges. A recent atomic force microscopic study has shown DNA nanoparticle formation in the presence of PAMAM dendrimers with some degree of uniformity (34). In our study, cellular uptake of ODN–dendrimer complexes was facile over a wide range of N/P ratios (ratio of the number of terminal amino groups in the dendrimer to the number of phosphate groups in the ODN). This is exemplified by the uptake of 32P-labeled ODN (0.4 nM) at an N/P ratio of 133 and of unlabeled ODN (0.25 μM) at an N/P ratio of 1, used for growth inhibition and western blot studies. The efficacy of uptake at this concentration of ODN was confirmed by mixing 32P-labeled ODN with unlabeled ODN and conducting the uptake studies (results not presented). However, the requirement for a high N/P ratio at the low ODN concentration may also indicate ion competition in the cell culture medium and the possibility that dendrimers may not be fully charged under these conditions (35) and/or steric hindrance in ODN–dendrimer interactions. These different possibilities will be addressed in future studies.
Results of PAGE experiments provide direct evidence for the increased stability of ODN in the presence of the dendrimer. Complexation with dendrimer markedly increased the availability of intact 32P-labeled ODN within the cells compared with uncomplexed 32P-labeled ODN. We tested the possible presence of ODN–G-4 clusters at the cell surface by washing the cells with DNase I solution. We found that a high level of ODN was retained in the cellular extract even after this washing (Fig. 6). This finding is important since the therapeutic success of gene delivery depends on the availability and retention of intact DNA within the cell for prolonged periods of time. The concentration of ODN required for inhibition of DNA synthesis decreased when ODN was complexed with G-4 dendrimer. In contrast, an ODN with reversed orientation did not cause a significant growth inhibition at 24, 48 or 72 h of treatment (Fig. 8B). In addition, western blot analysis showed a significant 65% decrease in c-myc protein level in cells treated with the triplex-forming oligonucleotide and G-4 (Fig. 9). Although these results are not sufficient proof for a triplex DNA mechanism, collectively they support the hypothesis that the triplex-forming oligonucleotide can exert sequence specificity in cell growth inhibition and c-myc protein expression.
A recent study by Zinselmeyer et al. (21) shows that PPI dendrimers can be used for the delivery of a plasmid DNA. In contrast to our observation, their report showed maximal uptake with G-2 dendrimer. The greater conformational mobility of the long DNA molecule may facilitate its interaction with the extended structure of lower generation dendrimers more efficiently than the interaction of these dendrimers with the 31 nt ODN used in our study. They also found that the number of binding sites between DNA and the dendrimer increased with molecular weight. It is possible that for a long DNA molecule, sufficient binding sites may be achieved with lower generation dendrimers compared with small ODNs. Thus, the size of the ODN/DNA might be an important factor in optimizing the efficiency of the dendrimer.
Uptake enhancement of ODN by dendrimers is perceived to be a consequence of their ability to form tight complexes with the ODN (36). The nanoscopic structures may pass through the lipid cellular membrane more easily than large particles. Because of the high local functional group and compact structure, the dendrimer can incorporate a large number of ODN on the surface and also interior tertiary amines. Evans et al. (36) recently reported structural polymorphism of DNA–dendrimer complexes using synchrotron X-ray diffraction and polarizing microscopic studies. An analogous situation is our recent findings of multiple liquid crystalline textures in polyamine-complexed calf thymus DNA (37). In our studies, the PPI dendrimer was found to condense the ODN, a phenomenon more commonly investigated with low molecular weight cations, such as spermine (1,25).
Zinselmeyer et al. (21) showed that the biocompatibility and transfection activity of G-2 and G-3 dendrimers were equivalent to commercial DOTAP. The size of the DNA–dendrimer complex determined by photon correlation spectroscopy in their experiment was between 163 and 518 nm. Data obtained for the ODN–dendrimer complex by dynamic light scattering experiments in our study showed a size of 130–280 nm, indicating that the size of the complex is not much affected by the chain length of ODN/DNA. No aggregation was observed at the physiologically compatible buffer conditions with 130 mM Na+/K+ ions, as particle size measurement was conducted in this buffer. The size of ODN nanoparticles was similar in a buffer containing 150 mM NaCl (unpublished results).
Haensler and Sz?ka (19) proposed that dendrimers with surface and internal amino groups can act as endosomal buffering agents similar to PEI, and can cause endosomal rupture, releasing the complex to the cytoplasm. Tang and Sz?ka (38) hypothesized that uncomplexed dendrimers adopt an extended conformation because of the repulsion between the charged surface amino groups. On complexation, the dendrimer collapses to a compact form because of charge neutralization (37). In the endosomal compartment, the interior tertiary amine becomes protonated, and hence less polymer is required to maintain charge neutralization. Excess polymer is then released and swells, causing endosomal rupture and release of the complex into the cytoplasm. The therapeutic efficacy of ODN depends on the intracellular fate and availability of ODN for hybridization with the target site. This can occur only when the ODN is free or loosely bound to the vector. Yoo and Juliano (39) found that unlike cationic lipid delivery agents, where the separation of the nucleic acid and delivery agent occurs in endosomes, the dendrimer–ODN complex remains intact as it enters the nucleus. Belinska et al. (40) reported that binding to dendrimer does not interfere with the ability of DNA to form hydrogen bonds with the complementary sequence in the target RNA or gene and that ODN detachment from the dendrimer is not essential for its action.
A major concern with the use of dendrimers as vectors for ODN delivery is their toxicity. The cytotoxicity of cationic dendrimers is believed to be due to the interaction between the positively charged dendrimer and negatively charged cellular structures (41,42). Malik et al. (42) reported the in vitro biocompatibility of G-2, G-3 and G-4 PPI dendrimers. The cytotoxicity measured by them showed an IC50 value of 0.3 mg/ml for B16F10 cells by MTT assay for G-2, 0.05 mg/ml for G-3, and 0.05 mg/ml for G-4 dendrimers. Lim et al. (43) reported that the cytotoxicity of PPI dendrimer is lower than that of PEI in Vero cells by MTT assay. The cell viability in our studies showed no toxicity for dendrimers of generations 1 to 5 under the concentrations used in ODN uptake and cell proliferation studies. Zinselmeyer et al. (21) found molecular weight-dependent toxicity by the MTT assay against a human epidermoid carcinoma cell line. In addition, Shah et al (44) found that the toxicity of dendrimers was reduced on complexation with DNA. They constructed an amphipathic asymmetric dendrimer and studied its transfection to BHK-21 cells in a complex with plasmid DNA. BHK cells were found to exhibit greater survival in the presence of the dendrimer–DNA complex than when dendrimer was applied alone. ODN–dendrimer complex has been shown to be less toxic than unmodified ODN, possibly due to the reduced charge of the complex (39,45). PPI dendrimer–gadolinium (Gd) complex has been reported to be useful as a magnetic resonance contrast agent (46,47). Toxicological studies of PPI G-5 dendrimer–Gd complex in mouse showed no significant adverse effect on body weight and organs compared with control. The mouse was injected with 0.15 mM of Gd/kg of the complex within 10 weeks (47).
Conclusions
In summary, this study shows that PPI dendrimers are highly efficient in enhancing the uptake of ODN by cells at non-toxic concentrations. Differences in efficiency with increasing generations of dendrimers suggest that the increased uptake is dependent on the charge density and flexibility of the dendrimer. Charge density increases and flexibility decreases with the increasing generation of the dendrimer. Optimal efficiency is obtained with G-4 dendrimer for the 31 nt ODN. The enhanced availability of intact ODN within cells up to 48 h or more and the improved antiproliferative effect of ODN on complexation with this dendrimer have important implications in ODN-based therapeutics. The high surface charge density of the polymer facilitates chemical modification. Assembling biologically interesting molecules such as amino acids, folate receptors, sugar derivatives, etc. at the dendrimer periphery presents an attractive avenue of research to improve the properties of this carrier. The versatility of its properties, ease of chemical modification and high cellular uptake of the complex by different cancer cell lines make PPI dendrimers an important new entry as an ODN/DNA delivery vehicle for gene therapy.
ACKNOWLEDGEMENTS
This work was supported, in part, by Public Health Service grants CA80163, CA73058 and CA42439 from the National Cancer Institute, and by a Susan G. Komen Breast Cancer Research Foundation Grant Award.
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