pH-responsive polymeric sirna carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1

Abstract

Small interfering RNA (siRNA)-based therapies have great potential for the treatment of debilitating diseases such as cancer, but an effective delivery strategy for siRNA is elusive. Here, pH-responsive complexes were developed for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells (NCI/ADR-RES) to doxorubicin. The electrostatic complexes consisted of a cationic micelle used as a nucleating core, siRNA, and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock copolymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly(styrene-alt-maleic anhydride) (pSMA) was electrostatically complexed with the positively charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30-105 nm) and charge (slightly positive) properties important for endocytosis and were found to be noncytotoxic and mediate uptake in >70% of ovarian cancer cells after 1 h of incubation. The pH-responsive ternary complexes were used to deliver siRNA against polo-like kinase 1 (plk1), a gene upregulated in many cancers and responsible for cell cycle progression, to ovarian cancer cell lines. Treatment resulted in approximately 50% reduction of plk1 gene expression in the drug-resistant NCI/ADR-RES ovarian cancer cell model and in the drug-sensitive parental cell line, OVCAR8. This knockdown functionally sensitized NCI/ADR-RES cells to doxorubicin at levels similar to OVCAR8. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following plk1 knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with doxorubicin and utilized in ternary complexes to achieve cell sensitization through simultaneous siRNA and drug delivery from a single carrier. These results show knockdown of plk1 results in sensitization of multidrug resistant cells to doxorubicin, and this combination of gene silencing and small molecule drug delivery may prove useful to achieve potent therapeutic effects.

Figure 1. Ternary complex formation and characterization

(A) Depiction of ternary complex formation including micelle charge ratio and overall charge ratio and (B) ternary complex characterization with respect to size and surface charge. (A) The diblock pDbB is shown in purple for poly(DMAEMA) and black for poly(butyl methacrylate), and siRNA is depicted in red. The pH-responsive polymer pSMA is shown in blue. Two charge ratios govern complex formation. The micelle charge ratio is defined as the ratio of positively-charged DMAEMA residues in the pDbB micelle corona to negatively-charged phosphate groups in the siRNA backbone. Based on published reports of the pDMAEMA pKa , the degree of protonation of the pDMAEMA corona was assumed to be 50%. The overall charge ratio accounts for the ratio of positive charges from the pDbB micelles to the total negative charges present from both siRNA and pSMA, based on the previously published pKa values for pSMA. (B) Micelles, micelles+siRNA, and ternary complexes were formed at various micellar and overall charge ratios at 25 nM siRNA concentrations in phosphate buffered saline and characterized for size and zeta potential using a ZetaPALS detector. Data are from three independent experiments conducted in triplicate.

Figure 2. Ternary complex cytotoxicity as a function of ternary carrier composition

NCI/ADR-RES cells were treated with siRNA (Negative control #1) at 25 nM using ternary complexes at various micelle charge ratios (listed first) and overall charge ratios (listed second). After 24 hours, cell lysate was collected and assayed for lactate dehydrogenase, a measure of cell viability, and data is shown relative to untreated cells. Data are from three independent experiments conducted in triplicate with error bars representing standard deviation. Statistical significance was evaluated at a level of p < 0.05 with the following symbols * indicating significance versus HiPerFect.

Figure 3. Ternary complex mediated siRNA uptake and plk1 knockdown

FAM-labeled siRNA uptake by NCI/ADR-RES cells was measured using flow cytometry 1 hour after treatment (A). Plk1 knockdown in OVCAR8 (black) and NCI/ADR-RES (white) cells was measured using real time RT-PCR 48 hours after treatment with ternary complexes as a function of micelle charge ratio with overall charge ratio fixed at 1.3:1 (B), and siRNA dose (5–50 nM). A commercially available transfection reagent, HiPerFect (Qiagen), was used as a positive control. For A, statistical significance was evaluated at a level of p < 0.05 with the following symbols indicating significance versus HiPerFect and treatments with micelle charge ratios of 2:1 and overall charge ratios of 1.3:1, respectively: *, #. For B, statistical significance was evaluated at a level of p < 0.05 with the following symbols indicating significance versus treatments of HiPerFect and micelle charge ratios of 8:1, respectively: *, #. For C, statistical significance was evaluated at a level of p < 0.05 with the following symbols indicating statistical significance versus treatments of 50 nM siRNA with HiPerFect, 50 nM siRNA with the ternary delivery system, and 25 nM siRNA with the ternary delivery system, respectively: *, #, and &.

Figure 4. plk1 knockdown sensitizes multidrug resistant cells to doxorubicin through p53 signaling

OVCAR8 and NCI/ADR-RES were treated with plk1 siRNA ternary complexes and doxorubicin (--□--; OVCAR8 (no doxorubicin), –■–; OVCAR8 + doxorubicin, --∆--; NCI/ADR-RES (no doxorubicin), –▲–; NCI/ADR-RES + doxorubicin. Resulting % viable cells (A) and caspase 3/7 upregulation (B) was assessed and expressed compared to untreated cells (as a percent in A or as a fold-upregulation in B). IC₅₀ values were extrapolated from A and listed in C. In addition, a p53 inhibitor, pifithrin α was added to the cells in addition to doxorubicin and resulting IC₅₀ values from those experiments are listed in C. Representative data ± standard deviations are shown from 3 independent studies done in quadruplicate. Statistical significance was evalulated at a level of p < 0.05 with * indicating significance versus cells without siRNA delivery at the same doxorubicin concentrations. IC₅₀ values were extrapolated from A and listed in C. In addition, a p53 inhibitor, pifithrin α was added to the cells in addition to doxorubicin and resulting IC₅₀ values from those experiments are listed in C. Representative data ± standard deviations are shown from 3 independent studies done in quadruplicate.

Figure 5. Doxorubicin-loaded ternary complexes are effective at initiating loss of cell viability and upregulated caspase 3/7 levels

OVCAR8 (black bars) and NCI/ADR-RES (white bars) were treated with dually-loaded plk1 siRNA and doxorubicin ternary complexes. Resulting cell viability (A) and caspase 3/7 upregulation (B) was assessed and expressed compared to untreated cells (as a percent in A or as a fold-upregulation in B). The doxorubicin dose was 0.2 µg/mL across treatments and experimental conditions. Statistical significance was tested, however there were no differences p > 0.05 between the two treatments, ternary complex-delivered plk1 siRNA and soluble doxorubicin and doxorubicin-loaded ternary complex delivered plk1 siRNA.

Scheme 1. Polymer Synthesis

(A) Synthesis of the diblock polymer pDbB via a two step RAFT polymerization. (B) A pH-sensitive derivative of pSMA was obtained by RAFT polymerization followed by subsequent reactions to modify 60% of the anhydride groups with pentylamine. R = CCNCH₃C₂H₄CO₂H, Z = SCSSC₁₂H₂₅, x ∼ 46, y ∼ 15 (∼ 31 for Figure 5), Z’ = SCSC₆H₅ n ∼ 190.