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Cationic polymer-mediated small interfering RNA delivery for P-glycoprotein down-regulation in tumor cells†
Article first published online: 16 AUG 2010
Copyright © 2010 American Cancer Society
Volume 116, Issue 23, pages 5544–5554, 1 December 2010
How to Cite
Abbasi, M., Lavasanifar, A., Berthiaume, L. G., Weinfeld, M. and Uludağ, H. (2010), Cationic polymer-mediated small interfering RNA delivery for P-glycoprotein down-regulation in tumor cells. Cancer, 116: 5544–5554. doi: 10.1002/cncr.25321
We thank Ms. V. Incani for providing the lipid-substituted polymers and Dr. R. Clarke for the cell line used for this study.
- Issue published online: 23 NOV 2010
- Article first published online: 16 AUG 2010
- Manuscript Accepted: 9 FEB 2010
- Manuscript Received: 3 NOV 2009
- permeability glycoprotein;
- small interfering RNA;
- multidrug resistance;
- polymeric carriers;
Among the treatment options that have been developed for cancer, chemotherapy remains 1 of the leading clinical approaches. Chemotherapy can usually control tumor growth at the onset of disease, but its effectiveness becomes limited by the overexpression of transporter proteins responsible for drug efflux, leading to multidrug resistance (MDR). To overcome this obstacle, the authors explored the feasibility of down-regulating the main drug transporter, P-glycoprotein (P-gp), by using nonviral small interfering RNA (siRNA) delivery as means to enhance the accumulation of chemotherapeutic agents in drug-resistant cancer cells.
Several cationic carriers capable of siRNA complexation were investigated for P-gp down-regulation in the MDA435/LCC6 cell line and, consequently, increased cellular uptake of the chemotherapeutic agents doxorubicin and paclitaxel.
Efficient siRNA delivery into tumor cells was demonstrated particularly using a palmitic-acid substituted poly(L-lysine), with no apparent differences in siRNA delivery between the wild type (WT)-expressing and P-gp-expressing phenotype (MDR1) of the cells. Efficient siRNA delivery led to approximately 40% to 50% P-gp suppression (based on the average expression level of the protein), an approximately 3-fold increased DOX uptake, and increased cytotoxicity in MDR1 cells.
The authors concluded that effective siRNA delivery with nonviral carriers can reduce the level of P-gp on cell surfaces and enhance the efficiency of chemotherapeutic agents in vitro. Cancer 2010. © 2010 American Cancer Society.
Cancer is among the leading causes of mortality worldwide. An estimated 1.7 million individuals will be diagnosed with cancer in 2009 in North America, and approximately 640,000 individuals will die of the disease.1, 2 Chemotherapy remains 1 of the main treatment options for patients with cancer, although its effectiveness can be limited by multidrug resistance (MDR).3, 4 MDR is caused by overexpression of cell membrane transporters, among which the P-glycoprotein (P-gp) has been identified as the most common form.5 P-gp is a member of the ABC-transporter family that reportedly is responsible for MDR in breast6, 7 and ovarian cancer cells.8 P-gp acts as an adenosine triphosphate (ATP)-dependant efflux pump that exports cytotoxic substrates of the cells. To increase drug uptake in tumor cells with high P-gp content, this transporter protein can be suppressed to increase the efficiency of chemotherapy. Several types of chemical inhibitors and modulators of P-gp activity have been explored9; however, to date, the clinical use of such chemical inhibitors has not led to clear success in reversing MDR.10
Unlike chemical regulators, RNA interference (RNAi) technology, in particular, small interfering RNAs (siRNAs),11 may provide a more effective approach for the down-regulation of protein targets like P-gp. The siRNAs usually are short molecules (21-25 nucleotides long) that are generated endogenously through the breakdown of long, double-stranded RNAs by dicers. Then, the siRNA is activated through binding to an RNA-induced silencing complex (RISC),12 which unwinds the siRNA duplex and produces an oligonucleotide specific for a target messenger RNA (mRNA), leading to cleavage and disposal of the resulting double-stranded RNA.13, 14 Among the advantages of using siRNA are its reduced toxicity on nonspecific tissues compared with conventional P-gp inhibitors and its high degree of specificity on a desired molecular target.15 The main obstacles for therapeutic use of siRNA are difficulty in transport through the cellular plasma membrane and the presence of nucleases in the physiologic milieu. siRNA cannot cross the lipid-bilayer plasma membrane on its own because of its anionic nature and requires a carrier for transport across the plasma membrane. Several groups have explored the feasibility of siRNA to suppress the MDR1 gene. Strong down-regulation of P-gp was obtained previously using a self-complementary recombinant adeno-associated virus (scAAV)16 in which approximately 80% P-gp protein suppression was achieved. However, significant concerns associated with viral carriers, such as the possibility of oncogenicity,17 inflammation of the target tissues,18 and cellular immune responses against viruses,19 will impede the translation of this approach into clinical practice. Nonviral delivery approaches could be more feasible in a clinical setting given their suitability for scale-up and their minimal, if any, immunogenicity. Nonviral siRNA delivery using lipid-based carriers has resulted in significant P-gp suppression in some reports6, 7, 20; however, a clear functional outcome, namely, increased intracellular accumulation and cytotoxic effect of drugs, has not been clearly demonstrated. Moreover, these carriers are intended for siRNA delivery in culture and display relatively weak protection of siRNA against serum21; thus, they are not suitable for systemic administration.
In a previous study, we demonstrated that lipid-modified polymers are efficient gene carriers that are able to condense relatively large plasmid DNAs (>1000 base pairs) and protect them against nuclease degradation.22, 23 Lipid substitution on cationic polymers enhanced the transport of plasmid DNA across cellular membranes.23 In the current study, we explored the feasibility of this type of carriers for siRNA delivery with the purpose of P-gp down-regulation in a tumor cell model. The ultimate objectives were to suppress the transporter activity responsible for chemoresistance and to increase drug concentrations in tumor cells, leading to increased cytotoxicity by the chemotherapeutic agents.8 The drugs that we included in this study are in current clinical use: doxorubicin (DOX) and paclitaxel (PTX).24 The uptake of these drugs into P-gp–expressing MDA435/LCC6 (MDR1) cells was assessed and compared with the drug uptake by the wild type (WT) cells. Drug uptake was assessed with or without siRNA inhibition of P-gp, and its consequences on drug toxicity were evaluated.
MATERIALS AND METHODS
Poly(L-lysine) (PLL) (25,500 daltons [Da]), polyethylenimine (PEI) (branched; 25,000 Da), and anhydrous dimethyl sulfoxide (DMSO) were purchased from Aldrich (Milwaukee, Wis). Hanks Balanced Salt Solution (HBSS), trypsin/ethylene diamine tetraacetic acid (EDTA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), cyclosporine A (CyA), and protease inhibitor cocktail were obtained from Sigma Chemical Company (St. Louis, Mo). Clear HBSS (phenol red free) was prepared in house. RPMI 1640 medium, penicillin (10,000 U/mL), and streptomycin (10 mg/mL) were obtained from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was obtained from PAA Laboratories Inc. (Etobicoke, Ontario). Fluorescence (FAM)-labeled siRNA was purchased from Ambion (Austin, Tex). Fluorescein isothiocyanate (FITC)-labeled mouse antihuman P-gp antibody was purchased from BD Biosciences Pharmingen (San Diego, Calif). The ABCB1 siRNA against P-gp (catalog no. SI00018732) and a control siRNA were purchased from QIAGEN (Mississauga, Ontario). DOX HCl was obtained from Fluka Analytical (Mumbai, India), and PTX was obtained from LC Laboratories (Woburn, Mass). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, Calif). WT MDA-435/LCC6 cells (referred to as WT cells) and their MDR1-transfected phenotype (referred as MDR1 cells) kindly were provided by Dr. R. Clarke (Georgetown University Medical School, Washington, DC). A lysis buffer was prepared by mixing 0.1% Tween in 50 mM Tris-Cl and 150 mM NaCl.
Carriers Used for siRNA Delivery
The carriers used for siRNA delivery were PLL, stearic acid-substituted PLL (PLL-StA), PEI, oleic acid-substituted PEI (PEI-OA), and Lipofectamine 2000. The synthesis and characterization of the PLL-StA (degree of substitution, 10 stearic acids per PLL) were described previously by Abbasi et al.22 The synthesis and characterization of PEI-OA (degree of substitution, 4.6 oleic acids per PEI) were described previously by Alshamsan et al.21
MTT Assay for Cytotoxicity
MDR1 cells and WT cells were seeded in 24-well plates in 0.5 mL RPMI medium and allowed to attach overnight. The medium was then aspirated, and 0.25 mL of fresh medium was added to the cells. Multiple concentrations of DOX and PTX ranging from 0.003 μg/mL to 19 μg/mL were incubated with the cells (in triplicate) for 24 hours. At the end of the incubation period, 20 μL of MTT stock solution in phosphate-buffered saline (5 mg/mL) were added to each well. After 3 hours, the medium was aspirated, and the precipitated formazan was dissolved in 200 μL DMSO. Cell viability was determined by measuring the absorbance (A) at 570 nm (with reference value at 650 nm). Cell viability was calculated as a percentage of control (untreated) cells as follows: ([A]test/[A]control) × 100%.
The MTT assay also was used to detect carrier and siRNA complex toxicities on the cells. To assess complex toxicities, the siRNA/carrier complexes were prepared at 1:10 siRNA:carrier ratios and were added to the cells for 24 hours at carrier concentrations of 0.425 μg/mL, 0.85 μg/mL, 1.75 μg/mL, and 3.5 μg/mL. The negative control siRNA was used to assess general toxicities of the siRNA complexes. The ABCB1 siRNA against P-gp was also used to investigate the effect of P-gp down-regulation on drug toxicities. For the latter investigations, the cells were treated with siRNA/carrier complexes for 48 hours (fresh complexes were added after 24 hours); then, DOX or PTX was added to the cells at a dose of 0.2 μg/mL, 0.6 μg/mL, and 2 μg/mL for 24 hours; and the MTT assay was performed as described above.
DOX Uptake by Flow Cytometry
The MDR1 and WT cells were incubated with multiple concentrations of DOX ranging from 0.003 μg/mL to 19 μg/mL for 24 hours in 24-well plates with 0.25 mL of fresh medium per well. After removing the DOX-containing media, the cells were washed with clear HBSS, trypsinized, and suspended in HBSS plus 3.7% formalin. DOX uptake was quantified with a Beckman Coulter flow cytometer (Cell Lab Quanta; Beckman Coulter, Inc., Brea, Calif) using the FL-2 detection channel to assess DOX-positive cells (∼3000 events per sample). The instrument was calibrated so that the negative control sample (ie, nontreated cells) yielded 1% to 2% cells that were positive for DOX.
CyA Inhibition of P-gp
The MDR1 cells in 24-well plates (with 0.25 mL medium per well) were exposed to 5 μg/mL CyA and DOX concentrations of 0.2 μg/mL, 0.6 μg/mL, and 2 μg/mL to detect DOX accumulation with chemical inhibition of P-gp. The cells were prepared for flow cytometry, and DOX assessment was performed using the FL-2 channel as described above.
Cellular Uptake of siRNA by Flow Cytometry and Agarose Gel Electrophoresis
The MDR1 and WT cells in 24-well plates (0.25 mL fresh medium per well) were incubated with FAM-labeled, scrambled siRNA to assess siRNA uptake by flow cytometry. A final concentration of 0.35 μg/mL of siRNA was incubated with 3.5 μg/mL of carrier in 150 mM NaCl to form complexes for 30 minutes. Then, the complexes were added to the cells in triplicate. After 24 hours, the media containing the complexes were removed, and the cells were washed with clear HBSS, trypsinized, and suspended in HBSS with 3.7% formalin. The siRNA uptake was quantified by flow cytometry using the FL-1 detection channel as described above to assess the FAM-positive cells.
In parallel studies, siRNA uptake was assessed by extracting the siRNA from MDR1 and WT cells and determining the amount of intact siRNA by gel electrophoresis. For this assessment, the cells were treated with the FAM-labeled siRNA complexes for 24 hours as described above, then washed with HBSS, and recovered by trypsinization at the indicated time points. The cell pellets were lyzed with 40 μL lysis buffer with the addition of 10 μL protease inhibitor. Cells were then placed on a shaker for 30 minutes and the solutions obtained after cell lysis were treated with heparin (0.625%) for 20 minutes and with EDTA (0.06 mM) for 5 minutes. Four microliters of 6 × diluted loading buffer were added to the samples, and the samples were run on a 1.5% agarose gel (at 120 V for 35 minutes). For a reference standard, an equivalent amount of the FAM-labeled siRNA (ie, an amount equal to the total amount exposed to the cells) was run on the gel. FAM-labeled siRNA was detected with a Fuji FLA-5000 flatbed scanner (Fujifilm, Tokyo, Japan) using a blue laser diode (485 nm), and the percentage recovery was calculated based on the spot densitometry as follows: 100% × (free siRNA/control siRNA).
P-gp Down-Regulation With siRNA
The MDR1 and WT cells in 24-well plates (with 0.25 mL of medium) were incubated with an ABCB1 siRNA specific for P-gp down-regulation. First, 3.5 μg/mL PLL-StA, PEI, PEI-OA, or Lipofectamine 2000 were incubated with 0.35 μg/mL ABCB1 siRNA for 30 minutes to form complexes. Then, the complexes were added to the cells either once (after 24 hours), twice (after 24 hours and 48 hours), or 3 times (after 24 hours, 48 hours, and 72 hours) at a concentration of 20 nM. At indicated time points, 10 μL of FITC-labeled antihuman P-gp antibody were added to the cells. The cells were then washed with clear HBSS, trypsinized, and suspended in HBSS with 3.7% formalin. The amount of P-gp in cells was quantified with the Beckman Coulter flow cytometer (Cell Lab Quanta) using the FL-1 detection channel as described above.
DOX Uptake After siRNA Treatment
The complexes were prepared with 0.35 μg/mL siRNA and 3.5 μg/mL PLL-StA, PEI, PEI-OA, and Lipofectamine 2000 and added to the MDR1 cells in 24-well plates (with 0.25 mL medium) for 24 hours or 48 hours. In the 48-hour experiment, fresh complexes were prepared and added to the cells after 24 hours. Cells were incubated with 0.2 μg/mL, 0.6 μg/mL, and 2 μg/mL DOX on Day 2 to detect DOX uptake after P-gp suppression. After an additional 24 hours of incubation, the flow cytometry analysis was performed using the FL-2 channel as described above to detect DOX uptake.
Where indicated, the current results are summarized as the mean ± standard deviation (SD) of the indicated number of replicates. Variations between the group means were analyzed with Student t tests. The level of significance was set at α = .05.
Characterization of MDA435/LCC6 MDR1 and WT Cells
The sensitivity of the cells to DOX and PTX is illustrated in Figure 1. After 24 hours of DOX treatment, the MDR1 cells had greater viability compared with the WT cells. A precipitous drop in cell viability was observed at 0.3 μg/mL DOX for WT cells, unlike the MDR1 phenotype (Fig. 1A). Similar results were obtained after 72 hours of DOX treatment (Fig. 1B). With PTX treatment, the MDR1 cells again had greater viability compared with the WT cells after 24 hours of treatment, and a precipitous drop in viability was observed at ∼3 μg/mL (Fig. 1C). After 72 hours of PTX treatment, WT cells displayed more cytotoxicity compared with MDR1 cells, and a precipitous drop in viability was observed at ∼3 μg/mL (Fig. 1D).
DOX uptake by the cells was assessed as a function of DOX concentration (Fig. 2). No uptake was detected at a DOX concentration <0.03 μg/mL; at greater concentrations, a DOX concentration-dependent uptake was observed in both cell types. At 0.3 μg/mL, ∼20% of MDR1 cells displayed uptake, whereas ∼80% of WT cells displayed DOX uptake. Almost all cells of both types displayed uptake at >0.6 μg/mL DOX (Fig. 2A). The mean fluorescence of the cell population (which is representative of the amount of DOX in cells) clearly differed between MDR1 cells and WT cells, with the latter cells producing greater DOX uptake at all concentrations tested (Fig. 2B). The chemical P-gp inhibitor CyA was effective in increasing DOX uptake by MDR1 cells; between DOX concentrations of 0.2 mM and 2 mM, a drastic increase in DOX uptake was evident in the presence of CyA based on both the percentage of DOX-positive cells (Fig. 2C) and the mean DOX levels in the cell population (Fig. 2D). CyA yielded almost 100% DOX-positive cells and increased DOX uptake by 20-fold at the highest DOX concentration tested (2 μg/mL). PTX uptake was not assessed in this study, because the drug displays no fluorescence.
siRNA uptake with PLL-StA, PEI, PEI-OA, and Lipofectamine 2000 was investigated by flow cytometry and was evident with all carriers with no differences between WT cells and MDR1 cells. In both cell types, siRNA uptake reached a maximal value of 80% to 100% of cells in 8 hours and remained high after 24 hours. After 48 hours, PEI and PEI-OA displayed maximal values, whereas PLL-StA and Lipofectamine 2000 produced a significant decrease (∼45%). The uptake after 72 hours displayed a similar pattern with a slight decrease in values (Fig. 3A).
The accumulation of siRNA in MDR1 and WT cells was investigated by recovering the internalized siRNA at different time points and assessing it quantitatively by gel electrophoresis. There was significant difference in siRNA recovery between WT cells and MDR1 cells, and the maximum amount of siRNA was recovered at 8 to 24 hours. At these time points, PEI and PEI-OA had the greatest siRNA recovery (40%-45%), PLL-StA had 30% to 35% siRNA recovery, and Lipofectamine 2000 had 15% to 20% siRNA recovery. The amount of siRNA was reduced significantly at 48 hours and 72 hours, although PEI and PEI-OA had ∼20% siRNA recovery at these time points (Fig. 3B).
P-gp Suppression by siRNA
ABCB1 siRNA was delivered to MDR1 cells by PLL-StA, PEI, PEI-OA, and Lipofectamine 2000; and P-gp levels were assessed by flow cytometry. To investigate the kinetics of P-gp suppression, the cells were treated with siRNA for 3 days with a daily repeated dose of complexes. The siRNA delivered by PLL-StA produced ∼40% P-gp suppression at all times (P < .05 vs no carrier). The siRNA delivered by PEI and PEI-OA produced no P-gp suppression on Day 1, produced ∼35% suppression on Day 2 (P < .03 vs no carrier), and produced 20% suppression on Day 3 (P < .05 vs no carrier). With Lipofectamine 2000, ∼40% P-gp suppression was detected on Days 2 and 3 (P < .04), but no suppression was detected on Day 1 (Fig. 4).
DOX Uptake and Cytotoxicity After siRNA Delivery to Cells
DOX uptake after P-gp suppression was assessed by flow cytometry. There was an increase in DOX uptake of the ABCB1 siRNA-treated cells compared with the scrambled siRNA-treated cells (Fig. 5). When ABCB1 siRNA was delivered by PLL-StA, 1.3-fold to 2.5-fold increased DOX levels were obtained when MDR1 cells were incubated with 0.2 to 2.0 μg/mL DOX (P < .02). No significant increase in DOX uptake was detected when the ABCB1 siRNA was delivered with PEI or PEI-OA. Lipofectamine 2000-delivered siRNA also effectively increased DOX uptake by ∼2-fold at 0.6 μg/mL and 2.0 μg/mL DOX (Fig. 5).
The cytotoxicity of DOX and PTX subsequently was assessed upon P-gp suppression. After siRNA delivery by PLL-StA, a 30% differential decrease in cell viability was detected between ABCB1 siRNA and scrambled siRNA (P < .01). The siRNA delivery with PEI was not effective in enhancing DOX cytotoxicity. There was a ∼30% decrease in cell viability when ABCB1 siRNA was delivered by Lipofectamine 2000 and cells were exposed to 0.2 μg/mL DOX (P < .02). Exposure to 0.6 μg/mL DOX concentration produced a ∼15% decrease in cell viability (P < .05), and exposure to 2 μg/mL DOX did not produce a difference in cell viability for Lipofectamine 2000-delivered siRNA (Fig. 6A). Analogous results were obtained when cells were treated with PTX; 1) siRNA delivered by PLL-StA produced a ∼20% decrease in cell viability at the tested PTX concentrations (P < .04), 2) PEI and PEI-OA were not effective in increasing the cytotoxicity of PTX, and 3) Lipofectamine 2000 produced a ∼20% decrease in cell viability analogous to that produced by the PLL-StA carrier (P < .05) (Fig. 6B).
We demonstrated previously that palmitic, myristic, and stearic acid-substituted PLLs are efficient carriers of plasmid DNA delivery to mammalian cells.22, 23 The finding that an efficient siRNA delivery also has been obtained with these polymers opens a new possibility for the use of the lipid-substituted polymers in down-regulating aberrant genes. The limitations of chemotherapy because of MDR are well recognized,8 and siRNA knockdown of P-gp is a promising option for overcoming this problem. In addition to P-gp, other proteins are involved in MDR, such as MDR-associated proteins 1 and 2 (MRP1 and MRP2),25 lung resistance-related protein (LRP),26 and breast cancer resistance protein (BCRP).25 However, P-gp is the most consistent link to MDR in cancer cells,27, 28 making it the natural focus for silencing. To validate the cell model that was chosen for the current study, overexpression of the MDR1 gene product P-gp was demonstrated in MDA435/LCC6 cells; >90% of MDR1 cells were positive for P-gp (not shown), and the cells experienced lower drug cytotoxicities, as indicated by the lower DOX and PTX uptake in these cells and demonstrated directly in the case of DOX. Combined with the CyA-based enhancement of DOX uptake, all of these analyses provide comprehensive validation of the chosen cell model for the purpose of the current study.
All carriers were able to protect siRNA against serum degradation at 1:10 siRNA:carrier ratios, but only polymeric carriers protected the siRNA against degradation at reduced siRNA:carrier ratios (1:3 and 1:1; results not shown). The weak protection afforded by Lipofectamine 2000 was consistent with other reports that indicated the superior ability of polymers to protect siRNA against serum nucleases.21, 29 The lipid modification of PLL led to greater delivery of siRNA into the cells. PLL-StA enabled siRNA delivery to almost all cells (∼90% by flow cytometry) but produced a significant drop in siRNA levels after 48 hours. This delivery pattern was similar to the delivery pattern observed with Lipofectamine 2000. The gradual decrease in siRNA levels may reflect the release of siRNA in the cytosol, leading to a gradual degradation of the molecule with intracellular nucleases. Whereas up to 30% of the siRNA incubated with the cells was recovered after 8 to 24 hours, almost no siRNA was recovered from the cells after 48 hours, which provided an indication of the time frame for complete siRNA degradation in the cytosol with PLL-StA and Lipofectamine 2000 (Fig. 3). PEI and PEI-OA had good ability to deliver siRNA into cells and provided delivery to almost all cells (80%-95%) after 48 to 72 hours. The finding that siRNA uptake and intracellular kinetics were similar in WT and MDR1 cells indicated that P-gp overexpression did not affect siRNA uptake and/or efflux.
Our initial P-gp down-regulation results indicated that higher carrier:siRNA ratios were more effective in P-gp knockdown. This may be caused by the better encapsulation of siRNA and, thus, a longer half-life of siRNA inside the cells. This finding was consistent with the data in the literature indicating higher gene knockdown at higher carrier:siRNA ratios.30, 31 P-gp down-regulation using 20 nM siRNA in the current study was a significant achievement, because most studies have reported P-gp down-regulation at siRNA concentrations >100 nM.6, 29 The use of such a low siRNA concentration also is important for minimizing nonspecific (ie, off-target) siRNA activity.32-34 With multiple siRNA treatments over 24-hour intervals, ∼40% to 50% P-gp suppression was gained after 72 hours using PLL-StA and Lipofectamine 2000. PEI and PEI-OA were not efficient at 24 hours but had greater efficiency at 48 hours and 72 hours, albeit at a lower levels than the other 2 carriers (Fig. 4). Incomplete or slow dissociation of siRNA from these polymers may have been the reason for this result, because siRNA dissociation from its carriers is paramount for its binding to the RISC complex and manifests the subsequent silencing activity.
The ultimate goal of P-gp knockdown is to impede drug efflux and increase intracellular levels of drugs that are P-gp substrates. However, the treatment of cells with siRNA complexes alone led to autofluorescence, complicating the assessment of DOX uptake. This phenomenon depended on both the carrier and the carrier concentration and particularly manifested with the PEI complexes. Cell treatment with complexes that were prepared with nonspecific siRNA was necessary to account for this apparent artifact. It was evident that higher concentrations of PEI (>1.75 μg/mL) lead to cytotoxicity and autofluorescence that gave false indications of DOX uptake in flow cytometry. siRNA delivery by PLL-StA resulted in an up to 2.5-fold increase in DOX-positive MDR1 cells, whereas siRNA delivery by PEI did not result in a considerable increase in DOX-positive cells. siRNA delivery with Lipofectamine 2000 yielded a desirable increase in DOX uptake only at the highest concentration tested (Fig. 5). Complementary cytotoxicity studies with the MTT assay revealed increases of ∼35% and ∼25% in the cytotoxicity of DOX-treated cells and PTX-treated cells, respectively, after siRNA delivery by PLL-StA. There was a good consistency between the carrier efficiency to suppress P-gp and the drug cytotoxicities obtained. To eliminate any complication from cytotoxic drugs (which potentially could eliminate a subpopulation of cells that display high uptake), cellular uptake of the P-gp substrate rhodamine also was explored after P-gp down-regulation and yielded results similar to those produced by the DOX uptake studies (results not shown). The CyA-treated cells had much higher elevations in DOX uptake (>10-fold) in terms of both the percentage of cells that displayed DOX uptake and the absolute DOX levels compared with siRNA-treated cells, indicating that siRNA-meditated silencing could be enhanced further. However, the nonspecific toxicity associated with CyA35 prevents its clinical application as a P-gp suppressor.
Other investigators also have attempted to use P-gp suppression by siRNA to reverse MDR. By using the MCF-7 breast cancer cell line, Stierle et al observed up to 60% P-gp suppression using siRNA delivery with Oligofectamine. This led to a ∼20% increase in drug uptake in these cells.16 Others also reported successful down-regulation of P-gp in these cells, including ∼65% P-gp suppression using Oligofectamine36 and 60% to 80% suppression using Lipofectamine 2000.17 However, those 2 studies did not report on drug accumulation inside the cells, and it is not known whether P-gp suppression resulted in a functional outcome. By using coblock copolymers suitable for micellazation, our previous studies yielded 50% to 60% P-gp suppression in the same MDA435/LCC6/MDR1 cells after siRNA delivery.30 Finally, Zhang et al produced 30% to 55% P-gp suppression with Oligofectamine in various ovarian cancer cell lines, albeit without reporting on drug accumulation in cells.20 Our P-gp suppression results using lipid-modified polymers are comparable to data in the literature on nonviral delivery, because we were able to suppress P-gp by 50% to 60% using PLL-StA for siRNA delivery. Lipofectamine 2000 (a carrier similar to Oligofectamine) produced variable results in our hands for P-gp suppression and did not lead to a significant increase in drug uptake. It is noteworthy that siRNA delivery by PEI-OA did not result in improvements in the down-regulation of P-gp suppression in the current study. In a previous report, we obtained effective siRNA delivery against integrin αv in B16 melanoma cells with PEI-OA.21 Possible differences in cell types and the target gene could explain the differences between these 2 studies.
The current results demonstrated that using lipid-modified PLL for the delivery of gene-specific siRNAs is an effective approach for down-regulating specific molecular targets. This study demonstrated an effective accumulation of chemotherapeutic agents in cells after P-gp suppression and increased cytotoxicity of both DOX and PTX, 2 issues that have not been addressed clearly by other nonviral approaches to siRNA delivery. Therefore, as concern increases over the use of viral carriers in cancer therapy, lipid-substituted polymeric carriers may be safe delivery systems.
CONFLICT OF INTEREST DISCLOSURES
Financial support for this project was provided by the Natural Sciences and Engineering Research Council of Canada and Canadian Institutes of Health Research. Equipment support was provided by the Alberta Heritage Foundation for Medical Research.
- 1Canadian Cancer Society. Canadian Cancer Statistics. Toronto, Ontario, Canada: Canadian Cancer Society; 2009.
- 3American Cancer Society. (Available at: http://www.cancer. org.
- 24Doxorubicin and paclitaxel improve survival compared with fluorouracil, doxorubicin and cyclophosphamide for women with metastatic breast cancer [serial online]. Evid-Based Oncol. 2001; 2: 4: 202-204..
- 25RNA expression of breast cancer resistance protein, lung resistance-related protein, multidrug resistance-associated proteins 1 and 2, and multidrug resistance gene 1 in breast cancer: correlation with chemotherapeutic response. Clin Cancer Res. 2003; 9: 827-836., , , et al.